Fibrolase is a non-hemorrhagic snake venom zinc metalloproteinase (EC 3.4.24.72) from southern copperhead (Agkistrodon contortrix contortrix) that exhibits direct fibrinolytic activity. Unlike plasminogen activators, it directly cleaves fibrin and fibrinogen at specific X-Leu bonds without requiring plasminogen activation, making it a unique fibrinolytic enzyme. As a venom component, it functions as a toxin that disrupts hemostasis in envenomated prey by dissolving blood clots. The enzyme requires zinc for catalytic activity and was developed into a recombinant therapeutic (alfimeprase) for clinical applications, though phase III trials were ultimately unsuccessful.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0035821
modulation of process of another organism
|
IEA
GO_REF:0000108 |
ACCEPT |
Summary: This annotation correctly identifies fibrolase's role in inter-organism interaction through venom. As a venom component, fibrolase directly affects hemostasis in prey/victim organisms by degrading fibrin/fibrinogen, thus modulating coagulation processes in the target organism.
Reason: Fibrolase is a venom metalloproteinase that directly affects another organism's physiology by disrupting hemostasis through fibrinolytic activity. This is a core function as it represents the primary biological role of this venom component [PMID:1898066, PMID:1304358].
Supporting Evidence:
PMID:1898066
The enzyme exhibits direct fibrinolytic activity and does not activate plasminogen. In vitro studies revealed that fibrolase dissolves clots made either from purified fibrinogen or from whole blood.
PMID:1304358
This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom.
|
|
GO:0004222
metalloendopeptidase activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This annotation correctly identifies the molecular function as a metalloendopeptidase. Fibrolase is classified as EC 3.4.24.72, a zinc metalloproteinase that cleaves peptide bonds in fibrin/fibrinogen. The term accurately captures the enzymatic mechanism.
Reason: The term accurately describes the core catalytic activity - fibrolase is indeed a zinc-dependent metalloendopeptidase (EC 3.4.24.72) that hydrolyzes internal peptide bonds. This is supported by extensive biochemical characterization showing zinc dependency and specific cleavage patterns [PMID:1898066].
Supporting Evidence:
PMID:1898066
the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator). Metal analysis revealed 1 mol of zinc/mol of protein.
PMID:1898066
Study of cleavage site preference of the fibrinolytic enzyme using the oxidized B chain of insulin revealed that specificity is similar to other snake venom metalloproteinases with cleavage primarily directed to an X-Leu bond.
|
|
GO:0005576
extracellular region
|
IEA
GO_REF:0000044 |
ACCEPT |
Summary: Correct annotation - fibrolase is a secreted venom protein found in the extracellular space after envenomation. The protein is secreted from venom glands and acts extracellularly on fibrin/fibrinogen substrates.
Reason: Fibrolase is definitively a secreted protein that functions in the extracellular environment. It is produced by venom glands and secreted as part of the venom to act on extracellular targets (fibrin/fibrinogen in blood) [PMID:1304358, PMID:1898066].
Supporting Evidence:
PMID:1304358
fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom
PMID:1898066
A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom has been purified
|
|
GO:0005886
plasma membrane
|
IEA
GO_REF:0000118 |
REMOVE |
Summary: This annotation is incorrect. Fibrolase is a secreted venom protein that acts in the extracellular space, not a plasma membrane-associated protein. There is no evidence for membrane localization.
Reason: Fibrolase is a secreted soluble enzyme that functions in the extracellular environment (blood plasma) after envenomation. It has no transmembrane domains or membrane-association motifs, and all functional studies show it acting as a soluble enzyme on soluble or fibrin-bound substrates. The UniProt entry clearly states it is "Secreted" with no membrane association [PMID:1304358, PMID:1898066].
Supporting Evidence:
PMID:1898066
The enzyme, fibrolase, has a molecular weight of 23,000-24,000 and an isoelectric point of pH 6.8. It is composed of approximately 200 amino acids
PMID:1304358
Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891
|
|
GO:0006508
proteolysis
|
IEA
GO_REF:0000120 |
MODIFY |
Summary: Correct but could be more specific. Fibrolase performs proteolysis specifically on fibrin/fibrinogen substrates. A more specific term like 'venom-mediated fibrinolysis' (GO:0044484) would better capture the biological context.
Reason: While proteolysis is correct, it's too general for this specialized venom enzyme. Fibrolase specifically performs fibrinolytic proteolysis as part of venom action. The term 'venom-mediated fibrinolysis' (GO:0044484) more accurately describes the biological process [PMID:1898066].
Proposed replacements:
venom-mediated fibrinolysis
Supporting Evidence:
PMID:1898066
The enzyme exhibits direct fibrinolytic activity and does not activate plasminogen. In vitro studies revealed that fibrolase dissolves clots made either from purified fibrinogen or from whole blood.
PMID:1304358
a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom
|
|
GO:0008233
peptidase activity
|
IEA
GO_REF:0000043 |
REMOVE |
Summary: This term is correct but too general. Fibrolase is specifically a metalloendopeptidase with defined substrate specificity, not just any peptidase.
Reason: This overly broad term adds no information beyond the more specific 'metalloendopeptidase activity' (GO:0004222) already present. The general peptidase activity is redundant with the more specific metallopeptidase annotations and doesn't capture the enzyme's specific catalytic mechanism or substrate specificity [PMID:1898066].
Supporting Evidence:
PMID:1898066
the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator)
|
|
GO:0008237
metallopeptidase activity
|
IEA
GO_REF:0000120 |
REMOVE |
Summary: Correct annotation that captures the metal-dependent peptidase activity. However, this is somewhat redundant with the more specific 'metalloendopeptidase activity' (GO:0004222) annotation.
Reason: This term is redundant with the more specific 'metalloendopeptidase activity' (GO:0004222) which better describes the enzyme's function. Metalloendopeptidase is a child term of metallopeptidase, so keeping both is unnecessary. The more specific term provides more information about the cleavage pattern (internal bonds) [PMID:1898066].
Supporting Evidence:
PMID:1898066
Study of cleavage site preference of the fibrinolytic enzyme using the oxidized B chain of insulin revealed that specificity is similar to other snake venom metalloproteinases with cleavage primarily directed to an X-Leu bond.
|
|
GO:0016787
hydrolase activity
|
IEA
GO_REF:0000043 |
REMOVE |
Summary: This extremely broad term is technically correct but uninformative. Fibrolase has much more specific annotations that describe its actual function.
Reason: This is an overly general parent term that provides no useful information beyond the specific metalloendopeptidase activity already annotated. Hydrolase activity is an ancestor of peptidase activity, which itself is an ancestor of metalloendopeptidase activity. Keeping this broad term adds no value [PMID:1898066].
Supporting Evidence:
PMID:1898066
the enzyme is a metalloproteinase
|
|
GO:0046872
metal ion binding
|
IEA
GO_REF:0000043 |
MODIFY |
Summary: Correct but could be more specific. Fibrolase specifically binds zinc ions, not just any metal ion. The more specific term 'zinc ion binding' (GO:0008270) would be more accurate.
Reason: While metal ion binding is correct, fibrolase specifically requires zinc for catalytic activity. Metal analysis confirmed 1 mol of zinc per mol of protein, and the enzyme is inhibited by zinc-specific chelators. The term 'zinc ion binding' (GO:0008270) more precisely describes this molecular function [PMID:1898066].
Proposed replacements:
zinc ion binding
Supporting Evidence:
PMID:1898066
Metal analysis revealed 1 mol of zinc/mol of protein
PMID:1898066
the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator)
|
|
GO:0090729
toxin activity
|
IEA
GO_REF:0000043 |
ACCEPT |
Summary: Correct annotation. Fibrolase is a venom toxin that disrupts hemostasis in envenomated organisms through its fibrinolytic activity, representing a key component of copperhead venom's toxic effects.
Reason: Fibrolase is indeed a toxin - it's a venom component that causes pathological effects in envenomated organisms by disrupting normal hemostasis through direct fibrinolysis. This is a core function as it represents the protein's primary biological role in the context of envenomation [PMID:1304358, PMID:1898066].
Supporting Evidence:
PMID:1304358
fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom
PMID:1898066
A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom
|
|
GO:0044484
venom-mediated fibrinolysis
|
IEA | NEW |
Summary: Core function of fibrolase in venom-mediated fibrinolysis
Reason: Fibrolase specifically performs fibrinolytic proteolysis as part of venom action to dissolve blood clots. This biological process is core to its function but was missing from existing annotations.
Supporting Evidence:
PMID:1898066
A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom
|
Q: How does fibrolase achieve substrate specificity for fibrin and other extracellular matrix proteins?
Q: What role does fibrolase play in venom composition and prey immobilization?
Q: How has fibrolase evolved to optimize its proteolytic activity in different environmental conditions?
Q: What are the structural features that determine fibrolase enzymatic activity and stability?
Experiment: Enzyme kinetics analysis to characterize fibrolase substrate specificity and catalytic parameters
Experiment: Structural determination using X-ray crystallography to understand the molecular basis of fibrolase activity
Experiment: Venom composition analysis to study fibrolase expression and co-expressed proteins
Experiment: Phylogenetic analysis of fibrolase evolution across different snake species
Exported on March 22, 2026 at 02:35 AM
Organism: AGKCO
Sequence:
QQRFPQRYVQLVIVADHRMNTKYNGDSDKIRQWVHQIVNTINEIYRPLNIQFTLVGLEIWSNQDLITVTSVSHDTLASFGNWRETDLLRRQRHDNAQLLTAIDFDGDTVGLAYVGGMCQLKHSTGVIQDHSAINLLVALTMAHELGHNLGMNHDGNQCHCGANSCVMAAMLSDQPSKLFSDCSKKDYQTFLTVNNPQCILNKP
I begin by examining the InterPro architecture. The N-terminal two-thirds of the protein is densely annotated with metallopeptidase signatures: IPR024079 (Metallopeptidase, catalytic domain superfamily; residues 3–203) defines a zinc-dependent catalytic fold; IPR001590 (Peptidase M12B, ADAM/reprolysin; residues 7–203) and IPR034027 (Reprolysin domain, adamalysin-type; residues 7–201) together specify an ADAM/reprolysin catalytic core. The coextensive spans of these domains from residues ~3–203 indicate a single, compact catalytic module typical of membrane-tethered metalloproteases. This architecture encodes a catalytic HExxH…E motif that coordinates Zn2+ and activates water for peptide-bond hydrolysis, establishing a metalloendopeptidase chemistry.
From this catalytic architecture, the primary molecular function follows as metalloendopeptidase activity that depends on a bound zinc ion. The presence of the reprolysin/ADAM family catalytic fold causes endopeptidic cleavage of extracellular or juxtamembrane substrates, aligning with GO:0004222 metalloendopeptidase activity and GO:0008270 zinc ion binding.
The biological process emerges from what such proteolysis accomplishes in neural and endocrine contexts. An ADAM-like catalytic module at the cell surface typically processes bioactive precursors and modulates signaling by shedding ectodomains or activating/inactivating cues. In neurons and endocrine cells, this proteolysis can tune pathways that govern synaptic maturation and stress responses. Therefore, the enzyme contributes to proteolytic processing that influences nervous system function, consistent with GO:0007165 signal transduction and the broader developmental framework of GO:0007267 cell–cell signaling as it mediates protease-dependent modulation of intercellular communication.
The cellular component and localization are dictated by the domain arrangement and the single-pass membrane topology. The catalytic module sits N-terminal to a transmembrane helix and short cytosolic tail characteristic of type I membrane proteases; this arrangement places the active site extracellularly while the C-terminus anchors in the bilayer. Such topology dictates a membrane-associated state, captured by GO:0016021 integral component of membrane, and is congruent with the experimentally supported membrane localization.
Mechanistically, I hypothesize that the extracellular catalytic domain cleaves extracellular or membrane-proximal substrates to regulate signaling at the neuronal and endocrine surfaces. Likely partners include membrane-tethered substrates and co-receptors: integrins and adhesion complexes can be processed by ADAM-like folds; membrane metalloprotease cohorts (e.g., other membrane proteases) may assemble into sheddase hubs; and synaptic organizers (such as neurexins/neuroligins or amyloid precursor-like substrates) could be modulated to fine-tune neural circuitry and stress hormone pathways. Together, these interactions would couple zinc-dependent proteolysis to signaling circuits that coordinate neural development and stress adaptation.
A membrane-tethered zinc-dependent endopeptidase that presents an extracellular catalytic module and a single-pass membrane anchor to execute proteolysis at the cell surface. By cleaving membrane-proximal and extracellular substrates, it modulates signaling pathways that shape neural function and endocrine communication, thereby contributing to nervous system activity and stress-responsive pathways while operating as an integral membrane protein.
May play some role in neural development and neural activity or general endocrine function. May be involved in general stress response pathway.
IPR024079, homologous_superfamily) — residues 3-203IPR001590, domain) — residues 7-203IPR034027, domain) — residues 7-201Molecular Function: molecular_function (GO:0003674), binding (GO:0005488), catalytic activity (GO:0003824), hydrolase activity (GO:0016787), ion binding (GO:0043167), catalytic activity, acting on a protein (GO:0140096), peptidase activity (GO:0008233), cation binding (GO:0043169), metallopeptidase activity (GO:0008237), endopeptidase activity (GO:0004175), metal ion binding (GO:0046872), magnesium ion binding (GO:0000287), metalloendopeptidase activity (GO:0004222), calcium ion binding (GO:0005509)
Biological Process: biological_process (GO:0008150), metabolic process (GO:0008152), biological regulation (GO:0065007), regulation of biological process (GO:0050789), regulation of response to stimulus (GO:0048583), regulation of biological quality (GO:0065008), regulation of multicellular organismal process (GO:0051239), regulation of hemostasis (GO:1900046), organic substance metabolic process (GO:0071704), nitrogen compound metabolic process (GO:0006807), primary metabolic process (GO:0044238), regulation of coagulation (GO:0050818), regulation of response to external stimulus (GO:0032101), regulation of blood coagulation (GO:0030193), regulation of body fluid levels (GO:0050878), organonitrogen compound metabolic process (GO:1901564), protein metabolic process (GO:0019538), macromolecule metabolic process (GO:0043170), regulation of response to stress (GO:0080134), proteolysis (GO:0006508), regulation of response to wounding (GO:1903034), regulation of wound healing (GO:0061041)
Cellular Component: cellular_component (GO:0005575), cellular anatomical entity (GO:0110165), extracellular region (GO:0005576)
Generated by BioReason
Exported on March 22, 2026 at 02:35 AM
Organism: AGKCO
Sequence:
QQRFPQRYVQLVIVADHRMNTKYNGDSDKIRQWVHQIVNTINEIYRPLNIQFTLVGLEIWSNQDLITVTSVSHDTLASFGNWRETDLLRRQRHDNAQLLTAIDFDGDTVGLAYVGGMCQLKHSTGVIQDHSAINLLVALTMAHELGHNLGMNHDGNQCHCGANSCVMAAMLSDQPSKLFSDCSKKDYQTFLTVNNPQCILNKP
I begin by examining the InterPro architecture. The N-terminal two-thirds of the protein is densely annotated with metallopeptidase signatures: IPR024079 (Metallopeptidase, catalytic domain superfamily; residues 3–203) defines a zinc-dependent catalytic fold; IPR001590 (Peptidase M12B, ADAM/reprolysin; residues 7–203) and IPR034027 (Reprolysin domain, adamalysin-type; residues 7–201) together specify an ADAM/reprolysin catalytic core. The coextensive spans of these domains from residues ~3–203 indicate a single, compact catalytic module typical of membrane-tethered metalloproteases. This architecture encodes a catalytic HExxH…E motif that coordinates Zn2+ and activates water for peptide-bond hydrolysis, establishing a metalloendopeptidase chemistry.
From this catalytic architecture, the primary molecular function follows as metalloendopeptidase activity that depends on a bound zinc ion. The presence of the reprolysin/ADAM family catalytic fold causes endopeptidic cleavage of extracellular or juxtamembrane substrates, aligning with GO:0004222 metalloendopeptidase activity and GO:0008270 zinc ion binding.
The biological process emerges from what such proteolysis accomplishes in neural and endocrine contexts. An ADAM-like catalytic module at the cell surface typically processes bioactive precursors and modulates signaling by shedding ectodomains or activating/inactivating cues. In neurons and endocrine cells, this proteolysis can tune pathways that govern synaptic maturation and stress responses. Therefore, the enzyme contributes to proteolytic processing that influences nervous system function, consistent with GO:0007165 signal transduction and the broader developmental framework of GO:0007267 cell–cell signaling as it mediates protease-dependent modulation of intercellular communication.
The cellular component and localization are dictated by the domain arrangement and the single-pass membrane topology. The catalytic module sits N-terminal to a transmembrane helix and short cytosolic tail characteristic of type I membrane proteases; this arrangement places the active site extracellularly while the C-terminus anchors in the bilayer. Such topology dictates a membrane-associated state, captured by GO:0016021 integral component of membrane, and is congruent with the experimentally supported membrane localization.
Mechanistically, I hypothesize that the extracellular catalytic domain cleaves extracellular or membrane-proximal substrates to regulate signaling at the neuronal and endocrine surfaces. Likely partners include membrane-tethered substrates and co-receptors: integrins and adhesion complexes can be processed by ADAM-like folds; membrane metalloprotease cohorts (e.g., other membrane proteases) may assemble into sheddase hubs; and synaptic organizers (such as neurexins/neuroligins or amyloid precursor-like substrates) could be modulated to fine-tune neural circuitry and stress hormone pathways. Together, these interactions would couple zinc-dependent proteolysis to signaling circuits that coordinate neural development and stress adaptation.
A membrane-tethered zinc-dependent endopeptidase that presents an extracellular catalytic module and a single-pass membrane anchor to execute proteolysis at the cell surface. By cleaving membrane-proximal and extracellular substrates, it modulates signaling pathways that shape neural function and endocrine communication, thereby contributing to nervous system activity and stress-responsive pathways while operating as an integral membrane protein.
May play some role in neural development and neural activity or general endocrine function. May be involved in general stress response pathway.
IPR024079, homologous_superfamily) — residues 3-203IPR001590, domain) — residues 7-203IPR034027, domain) — residues 7-201Molecular Function: molecular_function (GO:0003674), binding (GO:0005488), catalytic activity (GO:0003824), hydrolase activity (GO:0016787), ion binding (GO:0043167), catalytic activity, acting on a protein (GO:0140096), peptidase activity (GO:0008233), cation binding (GO:0043169), metallopeptidase activity (GO:0008237), endopeptidase activity (GO:0004175), metal ion binding (GO:0046872), magnesium ion binding (GO:0000287), metalloendopeptidase activity (GO:0004222), calcium ion binding (GO:0005509)
Biological Process: biological_process (GO:0008150), metabolic process (GO:0008152), biological regulation (GO:0065007), regulation of biological process (GO:0050789), regulation of response to stimulus (GO:0048583), regulation of biological quality (GO:0065008), regulation of multicellular organismal process (GO:0051239), regulation of hemostasis (GO:1900046), organic substance metabolic process (GO:0071704), nitrogen compound metabolic process (GO:0006807), primary metabolic process (GO:0044238), regulation of coagulation (GO:0050818), regulation of response to external stimulus (GO:0032101), regulation of blood coagulation (GO:0030193), regulation of body fluid levels (GO:0050878), organonitrogen compound metabolic process (GO:1901564), protein metabolic process (GO:0019538), macromolecule metabolic process (GO:0043170), regulation of response to stress (GO:0080134), proteolysis (GO:0006508), regulation of response to wounding (GO:1903034), regulation of wound healing (GO:0061041)
Cellular Component: cellular_component (GO:0005575), cellular anatomical entity (GO:0110165), extracellular region (GO:0005576)
Generated by BioReason
Fibrolase is a snake venom metalloproteinase (SVMP) from Agkistrodon contortrix contortrix (southern copperhead) venom that exhibits direct fibrinolytic activity. This protein has been extensively studied for its potential as a therapeutic fibrinolytic agent and was developed into a recombinant analog called alfimeprase for clinical applications.
Fibrolase is a zinc metalloproteinase that belongs to the venom metalloproteinase (M12B) family, P-I subfamily PMID:1304358. The enzyme requires zinc for its catalytic activity and structural integrity PMID:1898066.
Unlike many other fibrinolytic agents, fibrolase exhibits direct fibrinolytic activity and does not require activation of plasminogen PMID:1898066. This direct action makes it particularly attractive as a therapeutic agent because it bypasses the complex plasminogen activation cascade.
Fibrolase demonstrates specific cleavage preferences characteristic of snake venom metalloproteinases. The enzyme shows preferential cleavage at X-Leu bonds PMID:1898066. Specific cleavage sites include 14-Ala-|-Leu-15 in insulin B chain and 413-Lys-|-Leu-414 in the alpha-chain of fibrinogen.
A remarkable feature of fibrolase is its lack of hemorrhagic activity, which distinguishes it from many other snake venom metalloproteinases PMID:1898066. This property makes it more suitable for therapeutic applications as it reduces the risk of bleeding complications.
Fibrolase shows no activity against a series of chromogenic p-nitroanilide substrates, indicating its selective substrate specificity PMID:1898066.
Alfimeprase is a recombinant analog of fibrolase that was developed for clinical use PMID:11369866. This recombinant version maintains the fibrinolytic properties of the native enzyme while being produced in a controlled manufacturing environment.
Alfimeprase was investigated as a treatment for various thrombotic conditions PMID:18632486. The agent received orphan drug designation from both the FDA and the European Medicines Agency for evaluation in acute peripheral arterial occlusions PMID:18632486.
One of the primary clinical applications studied was the treatment of central venous catheter (CVC) occlusions, which are common in cancer patients receiving chemotherapy PMID:18632486. Alfimeprase showed promise in dissolving these occlusive clots.
Despite initial promise, alfimeprase ultimately failed phase III clinical trials for the treatment of arterial occlusive disease and acute ischemic stroke. This represents a significant setback in the translation of this snake venom-derived therapeutic from bench to bedside.
The primary physiological substrate for fibrolase is fibrin/fibrinogen, where it cleaves specific peptide bonds to dissolve blood clots PMID:1898066. The enzyme can act on both purified fibrinogen and whole blood clots, demonstrating its effectiveness in complex biological environments.
Detailed analysis using insulin B chain as a model substrate revealed the enzyme's preference for leucine at the P1' position (the amino acid immediately C-terminal to the cleavage site) PMID:1898066.
Fibrolase is composed of 203 amino acid residues in a single polypeptide chain with a calculated molecular weight of 22,891 Da PMID:1304358. The N-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue PMID:1304358.
The protein contains six cysteine residues that form three disulfide bonds critical for structural stability PMID:1304358. The disulfide connectivity was definitively established for the recombinant analog alfimeprase as Cys-116/196, Cys-156/180, and Cys-158/163 PMID:11369866.
Structural analysis reveals that fibrolase has very low alpha-helical content (4%) but significant beta-structure (39.5%) PMID:1304358. This structural composition is consistent with its classification as a metalloproteinase.
The protein contains little or no carbohydrate and lacks asparagine-linked glycosylation sites PMID:1898066. Analysis revealed the absence of consensus N-glycosylation sequences (asparagine-X-serine/threonine) PMID:1304358.
Natural fibrolase exhibits microheterogeneity at both the N-terminus and at positions 189 and 192 PMID:1304358, suggesting natural variations in the venom extract.
As a zinc metalloproteinase, fibrolase is sensitive to metal chelators. The enzyme is inhibited by EDTA, o-phenanthroline, and tetraethylenepentamine (a specific zinc chelator) PMID:1898066. This sensitivity to metal chelators is consistent with zinc being essential for both catalytic activity and structural integrity.
Fibrolase is not inhibited by classical serine protease inhibitors including diisopropylfluorophosphate (DFP), soybean trypsin inhibitor, and Trasylol PMID:1898066. This resistance profile confirms its classification as a metalloproteinase rather than a serine protease.
The enzyme is not inhibited by p-chloromercuribenzoate, indicating that free thiol groups are not essential for its activity PMID:1898066, suggesting that the disulfide bonds provide sufficient structural stability.
The absolute requirement for zinc is demonstrated by the complete loss of activity upon zinc removal and the restoration of activity upon zinc repletion. Metal analysis confirmed that fibrolase contains exactly 1 mol of zinc per mol of protein PMID:1898066.
Fibrolase represents a unique approach to thrombolytic therapy due to several key advantages:
Direct Action: Unlike plasminogen activators such as tissue plasminogen activator (tPA), fibrolase acts directly on fibrin without requiring the plasminogen activation cascade PMID:1898066.
Non-hemorrhagic Profile: The lack of hemorrhagic activity reduces the risk of bleeding complications that are common with other thrombolytic agents PMID:1898066.
Specific Substrate Recognition: The enzyme shows selectivity for fibrin substrates over other proteins, potentially reducing off-target effects.
Despite its promising properties, the clinical development of alfimeprase faced significant challenges. The failure of phase III clinical trials highlights the complexity of translating promising preclinical results into successful clinical outcomes. Factors that may have contributed to clinical failure include:
Immunogenicity: As a foreign protein, alfimeprase may have triggered immune responses that limited its efficacy or safety.
Pharmacokinetics: The in vivo behavior of the recombinant protein may have differed from expectations based on in vitro studies.
Clinical Trial Design: The specific patient populations, dosing regimens, or outcome measures may not have been optimally designed to demonstrate efficacy.
The development of alfimeprase occurred during a period when the thrombolytic therapy field was seeking alternatives to urokinase, which had been withdrawn from the market due to manufacturing issues PMID:18632486. This created a clinical need for new fibrinolytic agents with improved safety profiles.
From a research perspective, fibrolase has provided important insights into:
Structure-Function Relationships: The detailed structural characterization has advanced understanding of metalloproteinase catalysis and specificity.
Venom Biology: Fibrolase represents an example of how evolution has optimized enzymes for specific biological functions.
Drug Development: The alfimeprase story illustrates both the potential and the challenges of developing venom-derived therapeutics.
Fibrolase is a fascinating example of a snake venom component that has been extensively characterized and developed for therapeutic applications. Its unique combination of direct fibrinolytic activity without hemorrhagic effects made it an attractive candidate for treating thrombotic conditions. While the clinical development of alfimeprase ultimately was unsuccessful, the extensive research on fibrolase has contributed significantly to our understanding of metalloproteinase structure and function, as well as the challenges of developing venom-derived therapeutics. The protein remains an important model system for studying fibrinolytic enzymes and continues to provide insights into the design of improved thrombolytic agents.
Source: fibrolase-deep-research-bioreason-rl.md
The BioReason summary states:
A membrane-tethered zinc-dependent endopeptidase that presents an extracellular catalytic module and a single-pass membrane anchor to execute proteolysis at the cell surface. By cleaving membrane-proximal and extracellular substrates, it modulates signaling pathways that shape neural function and endocrine communication, thereby contributing to nervous system activity and stress-responsive pathways while operating as an integral membrane protein.
This summary is fundamentally incorrect in multiple ways:
Not membrane-tethered: The summary describes fibrolase as a "membrane-tethered" "integral membrane protein." The curated review explicitly states fibrolase is a "secreted venom protein" localized to the extracellular region (GO:0005576). The curated review even removes the plasma membrane annotation (GO:0005886) as incorrect: "Fibrolase is a secreted soluble enzyme that functions in the extracellular environment... It has no transmembrane domains or membrane-association motifs."
Not involved in neural/endocrine function: The summary claims fibrolase "modulates signaling pathways that shape neural function and endocrine communication." This appears to be confabulated from the generic ADAM/reprolysin family biology. Fibrolase is a snake venom metalloproteinase that cleaves fibrin and fibrinogen -- its function is fibrinolytic, disrupting hemostasis in envenomated prey.
No mention of venom or fibrinolytic function: The curated review identifies fibrolase as a "non-hemorrhagic snake venom zinc metalloproteinase (EC 3.4.24.72) from southern copperhead that exhibits direct fibrinolytic activity." The core biological processes are venom-mediated fibrinolysis (GO:0044484) and toxin activity (GO:0090729). None of this appears in the BioReason summary.
Zinc metalloendopeptidase activity is the one correct element: BioReason correctly identifies the metalloendopeptidase catalytic mechanism with zinc dependency, matching GO:0004222 in the curated review.
Root cause: BioReason appears to have defaulted to the generic ADAM/reprolysin family functional template (membrane-anchored sheddase involved in neural/endocrine signaling) without recognizing that fibrolase is a secreted venom enzyme. The ADAM/reprolysin domain annotation (IPR001590) covers both membrane-bound ADAMs and secreted SVMPs (snake venom metalloproteinases), but BioReason assumed the membrane-bound model.
Comparison with interpro2go:
The ai-review.yaml does not contain GO_REF:0000002 annotations for fibrolase. However, some IEA annotations make errors analogous to BioReason's: GO_REF:0000118 (TreeGrafter) assigned plasma membrane (GO:0005886), which the curated review removes. BioReason's error of inferring membrane-tethered localization parallels this automated annotation error. Both are caused by the ADAM/reprolysin domain family encompassing both membrane-bound and secreted enzymes, with the automated methods defaulting to the more common membrane-bound variant.
The trace identifies the metallopeptidase catalytic domain and reprolysin/ADAM-type assignment correctly. However, it then infers "a single-pass membrane topology" and "type I membrane protease" from the ADAM family template, which is incorrect for this secreted venom protein. The trace also hypothesizes "neurexins/neuroligins or amyloid precursor-like substrates" as targets, reflecting ADAM biology rather than venom metalloproteinase biology.
id: P28891
gene_symbol: fibrolase
taxon:
id: NCBITaxon:8713
label: Agkistrodon contortrix contortrix
description: Fibrolase is a non-hemorrhagic snake venom zinc metalloproteinase (EC
3.4.24.72) from southern copperhead (Agkistrodon contortrix contortrix) that exhibits
direct fibrinolytic activity. Unlike plasminogen activators, it directly cleaves
fibrin and fibrinogen at specific X-Leu bonds without requiring plasminogen activation,
making it a unique fibrinolytic enzyme. As a venom component, it functions as a
toxin that disrupts hemostasis in envenomated prey by dissolving blood clots. The
enzyme requires zinc for catalytic activity and was developed into a recombinant
therapeutic (alfimeprase) for clinical applications, though phase III trials were
ultimately unsuccessful.
existing_annotations:
- term:
id: GO:0035821
label: modulation of process of another organism
evidence_type: IEA
original_reference_id: GO_REF:0000108
review:
summary: This annotation correctly identifies fibrolase's role in inter-organism
interaction through venom. As a venom component, fibrolase directly affects
hemostasis in prey/victim organisms by degrading fibrin/fibrinogen, thus modulating
coagulation processes in the target organism.
action: ACCEPT
reason: Fibrolase is a venom metalloproteinase that directly affects another organism's
physiology by disrupting hemostasis through fibrinolytic activity. This is a
core function as it represents the primary biological role of this venom component
[PMID:1898066, PMID:1304358].
supported_by:
- reference_id: PMID:1898066
supporting_text: The enzyme exhibits direct fibrinolytic activity and does not
activate plasminogen. In vitro studies revealed that fibrolase dissolves clots
made either from purified fibrinogen or from whole blood.
- reference_id: PMID:1304358
supporting_text: This is the first report of the sequence of a direct-acting,
nonhemorrhagic fibrinolytic enzyme found in snake venom.
- term:
id: GO:0004222
label: metalloendopeptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: This annotation correctly identifies the molecular function as a metalloendopeptidase.
Fibrolase is classified as EC 3.4.24.72, a zinc metalloproteinase that cleaves
peptide bonds in fibrin/fibrinogen. The term accurately captures the enzymatic
mechanism.
action: ACCEPT
reason: The term accurately describes the core catalytic activity - fibrolase
is indeed a zinc-dependent metalloendopeptidase (EC 3.4.24.72) that hydrolyzes
internal peptide bonds. This is supported by extensive biochemical characterization
showing zinc dependency and specific cleavage patterns [PMID:1898066].
supported_by:
- reference_id: PMID:1898066
supporting_text: the enzyme is a metalloproteinase since it is inhibited by
EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator).
Metal analysis revealed 1 mol of zinc/mol of protein.
- reference_id: PMID:1898066
supporting_text: Study of cleavage site preference of the fibrinolytic enzyme
using the oxidized B chain of insulin revealed that specificity is similar
to other snake venom metalloproteinases with cleavage primarily directed to
an X-Leu bond.
- term:
id: GO:0005576
label: extracellular region
evidence_type: IEA
original_reference_id: GO_REF:0000044
review:
summary: Correct annotation - fibrolase is a secreted venom protein found in the
extracellular space after envenomation. The protein is secreted from venom glands
and acts extracellularly on fibrin/fibrinogen substrates.
action: ACCEPT
reason: Fibrolase is definitively a secreted protein that functions in the extracellular
environment. It is produced by venom glands and secreted as part of the venom
to act on extracellular targets (fibrin/fibrinogen in blood) [PMID:1304358,
PMID:1898066].
supported_by:
- reference_id: PMID:1304358
supporting_text: fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon
contortrix contortrix) venom
- reference_id: PMID:1898066
supporting_text: A fibrinolytic enzyme present in Agkistrodon contortrix contortrix
(southern copperhead) venom has been purified
- term:
id: GO:0005886
label: plasma membrane
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: This annotation is incorrect. Fibrolase is a secreted venom protein that
acts in the extracellular space, not a plasma membrane-associated protein. There
is no evidence for membrane localization.
action: REMOVE
reason: Fibrolase is a secreted soluble enzyme that functions in the extracellular
environment (blood plasma) after envenomation. It has no transmembrane domains
or membrane-association motifs, and all functional studies show it acting as
a soluble enzyme on soluble or fibrin-bound substrates. The UniProt entry clearly
states it is "Secreted" with no membrane association [PMID:1304358, PMID:1898066].
supported_by:
- reference_id: PMID:1898066
supporting_text: The enzyme, fibrolase, has a molecular weight of 23,000-24,000
and an isoelectric point of pH 6.8. It is composed of approximately 200 amino
acids
- reference_id: PMID:1304358
supporting_text: Fibrolase is composed of 203 residues in a single polypeptide
chain with a molecular weight of 22,891
- term:
id: GO:0006508
label: proteolysis
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: Correct but could be more specific. Fibrolase performs proteolysis specifically
on fibrin/fibrinogen substrates. A more specific term like 'venom-mediated fibrinolysis'
(GO:0044484) would better capture the biological context.
action: MODIFY
reason: While proteolysis is correct, it's too general for this specialized venom
enzyme. Fibrolase specifically performs fibrinolytic proteolysis as part of
venom action. The term 'venom-mediated fibrinolysis' (GO:0044484) more accurately
describes the biological process [PMID:1898066].
proposed_replacement_terms:
- id: GO:0044484
label: venom-mediated fibrinolysis
supported_by:
- reference_id: PMID:1898066
supporting_text: The enzyme exhibits direct fibrinolytic activity and does not
activate plasminogen. In vitro studies revealed that fibrolase dissolves clots
made either from purified fibrinogen or from whole blood.
- reference_id: PMID:1304358
supporting_text: a direct-acting, nonhemorrhagic fibrinolytic enzyme found in
snake venom
- term:
id: GO:0008233
label: peptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: This term is correct but too general. Fibrolase is specifically a metalloendopeptidase
with defined substrate specificity, not just any peptidase.
action: REMOVE
reason: This overly broad term adds no information beyond the more specific 'metalloendopeptidase
activity' (GO:0004222) already present. The general peptidase activity is redundant
with the more specific metallopeptidase annotations and doesn't capture the
enzyme's specific catalytic mechanism or substrate specificity [PMID:1898066].
supported_by:
- reference_id: PMID:1898066
supporting_text: the enzyme is a metalloproteinase since it is inhibited by
EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator)
- term:
id: GO:0008237
label: metallopeptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: Correct annotation that captures the metal-dependent peptidase activity.
However, this is somewhat redundant with the more specific 'metalloendopeptidase
activity' (GO:0004222) annotation.
action: REMOVE
reason: This term is redundant with the more specific 'metalloendopeptidase activity'
(GO:0004222) which better describes the enzyme's function. Metalloendopeptidase
is a child term of metallopeptidase, so keeping both is unnecessary. The more
specific term provides more information about the cleavage pattern (internal
bonds) [PMID:1898066].
supported_by:
- reference_id: PMID:1898066
supporting_text: Study of cleavage site preference of the fibrinolytic enzyme
using the oxidized B chain of insulin revealed that specificity is similar
to other snake venom metalloproteinases with cleavage primarily directed to
an X-Leu bond.
- term:
id: GO:0016787
label: hydrolase activity
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: This extremely broad term is technically correct but uninformative. Fibrolase
has much more specific annotations that describe its actual function.
action: REMOVE
reason: This is an overly general parent term that provides no useful information
beyond the specific metalloendopeptidase activity already annotated. Hydrolase
activity is an ancestor of peptidase activity, which itself is an ancestor of
metalloendopeptidase activity. Keeping this broad term adds no value [PMID:1898066].
supported_by:
- reference_id: PMID:1898066
supporting_text: the enzyme is a metalloproteinase
- term:
id: GO:0046872
label: metal ion binding
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: Correct but could be more specific. Fibrolase specifically binds zinc
ions, not just any metal ion. The more specific term 'zinc ion binding' (GO:0008270)
would be more accurate.
action: MODIFY
reason: While metal ion binding is correct, fibrolase specifically requires zinc
for catalytic activity. Metal analysis confirmed 1 mol of zinc per mol of protein,
and the enzyme is inhibited by zinc-specific chelators. The term 'zinc ion binding'
(GO:0008270) more precisely describes this molecular function [PMID:1898066].
proposed_replacement_terms:
- id: GO:0008270
label: zinc ion binding
supported_by:
- reference_id: PMID:1898066
supporting_text: Metal analysis revealed 1 mol of zinc/mol of protein
- reference_id: PMID:1898066
supporting_text: the enzyme is a metalloproteinase since it is inhibited by
EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator)
- term:
id: GO:0090729
label: toxin activity
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: Correct annotation. Fibrolase is a venom toxin that disrupts hemostasis
in envenomated organisms through its fibrinolytic activity, representing a key
component of copperhead venom's toxic effects.
action: ACCEPT
reason: Fibrolase is indeed a toxin - it's a venom component that causes pathological
effects in envenomated organisms by disrupting normal hemostasis through direct
fibrinolysis. This is a core function as it represents the protein's primary
biological role in the context of envenomation [PMID:1304358, PMID:1898066].
supported_by:
- reference_id: PMID:1304358
supporting_text: fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon
contortrix contortrix) venom
- reference_id: PMID:1898066
supporting_text: A fibrinolytic enzyme present in Agkistrodon contortrix contortrix
(southern copperhead) venom
- term:
id: GO:0044484
label: venom-mediated fibrinolysis
evidence_type: IEA
review:
summary: Core function of fibrolase in venom-mediated fibrinolysis
action: NEW
reason: Fibrolase specifically performs fibrinolytic proteolysis as part of venom
action to dissolve blood clots. This biological process is core to its function
but was missing from existing annotations.
supported_by:
- reference_id: PMID:1898066
supporting_text: A fibrinolytic enzyme present in Agkistrodon contortrix contortrix
(southern copperhead) venom
core_functions:
- description: Zinc metalloendopeptidase that directly cleaves fibrin and fibrinogen
at X-Leu bonds to dissolve blood clots
molecular_function:
id: GO:0004222
label: metalloendopeptidase activity
directly_involved_in:
- id: GO:0044484
label: venom-mediated fibrinolysis
locations:
- id: GO:0005576
label: extracellular region
substrates:
- id: CHEBI:5054
label: Fibrin
- id: GO:0005577
label: fibrinogen complex
- description: Acts as a venom toxin that modulates hemostasis in envenomated organisms
molecular_function:
id: GO:0090729
label: toxin activity
directly_involved_in:
- id: GO:0035821
label: modulation of process of another organism
locations:
- id: GO:0005576
label: extracellular region
references:
- id: GO_REF:0000043
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
findings: []
- id: GO_REF:0000044
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
vocabulary mapping, accompanied by conservative changes to GO terms applied by
UniProt.
findings: []
- id: GO_REF:0000108
title: Automatic assignment of GO terms using logical inference, based on on inter-ontology
links.
findings: []
- id: GO_REF:0000118
title: TreeGrafter-generated GO annotations
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods.
findings: []
- id: PMID:1898066
title: Purification and characterization of a fibrinolytic enzyme from venom of
the southern copperhead snake (Agkistrodon contortrix contortrix).
findings:
- statement: Fibrolase is a zinc metalloproteinase containing 1 mol of zinc per
mol of protein
supporting_text: However, the enzyme is a metalloproteinase since it is inhibited
by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator).
Metal analysis revealed 1 mol of zinc/mol of protein.
full_text_unavailable: true
- statement: Fibrolase exhibits substrate specificity similar to other snake venom
metalloproteinases with preference for X-Leu bonds
supporting_text: Study of cleavage site preference of the fibrinolytic enzyme
using the oxidized B chain of insulin revealed that specificity is similar to
other snake venom metalloproteinases with cleavage primarily directed to an
X-Leu bond.
full_text_unavailable: true
- statement: Fibrolase exhibits direct fibrinolytic activity without activating
plasminogen
supporting_text: The enzyme exhibits direct fibrinolytic activity and does not
activate plasminogen. In vitro studies revealed that fibrolase dissolves clots
made either from purified fibrinogen or from whole blood.
full_text_unavailable: true
- statement: Fibrolase has molecular weight of 23,000-24,000 Da and isoelectric
point of pH 6.8
supporting_text: The enzyme, fibrolase, has a molecular weight of 23,000-24,000
and an isoelectric point of pH 6.8.
full_text_unavailable: true
- statement: Fibrolase is composed of approximately 200 amino acids with blocked
NH2-terminus and contains little or no carbohydrate
supporting_text: It is composed of approximately 200 amino acids, possesses a
blocked NH2-terminus and contains little or no carbohydrate.
full_text_unavailable: true
- statement: Unlike some other venom fibrinolytic metalloproteinases, fibrolase
exhibits little or no hemorrhagic activity
supporting_text: Interestingly, unlike some other venom fibrinolytic metalloproteinases,
fibrolase exhibits little if any hemorrhagic activity.
full_text_unavailable: true
- statement: Fibrolase is not inhibited by serine protease inhibitors but is specifically
inhibited by metal chelators
supporting_text: The enzyme shows no activity against a series of chromogenic
p-nitroanilide substrates and is not inhibited by diisopropylfluorophosphate,
soybean trypsin inhibitor, Trasylol, or p-chloromercuribenzoate. However, the
enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline
and tetraethylenepentamine (a specific zinc chelator).
full_text_unavailable: true
- id: PMID:1304358
title: Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from
Agkistrodon contortrix contortrix venom.
findings:
- statement: Fibrolase is the first sequenced direct-acting, nonhemorrhagic fibrinolytic
enzyme from snake venom
supporting_text: This is the first report of the sequence of a direct-acting,
nonhemorrhagic fibrinolytic enzyme found in snake venom.
full_text_unavailable: true
- statement: Fibrolase consists of 203 amino acid residues in a single polypeptide
chain with molecular weight of 22,891 Da
supporting_text: Fibrolase is composed of 203 residues in a single polypeptide
chain with a molecular weight of 22,891, as determined by the sequence.
full_text_unavailable: true
- statement: Fibrolase has a blocked amino-terminus with a pyroglutamic acid residue
supporting_text: The amino-terminus is blocked by a cyclized glutamine (pyroglutamic
acid) residue, and the sequence of this region of the molecule was determined
by mass spectrometry.
full_text_unavailable: true
- statement: Fibrolase shows sequence homology to hemorrhagic snake venom metalloproteinases
supporting_text: Its sequence is homologous to the sequence of the hemorrhagic
toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases
from Trimeresurus flavoviridis venom.
full_text_unavailable: true
- statement: Fibrolase contains six cysteine residues involved in three disulfide
bonds with specific connectivity pattern
supporting_text: All six cysteine residues in fibrolase are involved in disulfide
bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established
and bonds between cysteines-158/165 and between cysteines-160/192 are inferred
from the homology to Ht-d.
full_text_unavailable: true
- statement: Fibrolase has predominantly beta-sheet secondary structure with minimal
alpha-helix content
supporting_text: Secondary structure prediction reveals a very low percentage
of alpha-helix (4%), but much greater beta-structure (39.5%).
full_text_unavailable: true
- statement: Fibrolase lacks asparagine-linked glycosylation sites
supporting_text: 'Analysis of the sequence reveals the absence of asparagine-linked
glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.'
full_text_unavailable: true
- statement: Fibrolase exhibits microheterogeneity at the amino-terminus and at
specific residue positions
supporting_text: Microheterogeneity in the sequence was found at both the amino-terminus
and at residues 189 and 192.
full_text_unavailable: true
suggested_questions:
- question: How does fibrolase achieve substrate specificity for fibrin and other
extracellular matrix proteins?
- question: What role does fibrolase play in venom composition and prey immobilization?
- question: How has fibrolase evolved to optimize its proteolytic activity in different
environmental conditions?
- question: What are the structural features that determine fibrolase enzymatic activity
and stability?
suggested_experiments:
- description: Enzyme kinetics analysis to characterize fibrolase substrate specificity
and catalytic parameters
- description: Structural determination using X-ray crystallography to understand
the molecular basis of fibrolase activity
- description: Venom composition analysis to study fibrolase expression and co-expressed
proteins
- description: Phylogenetic analysis of fibrolase evolution across different snake
species
status: COMPLETE
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