Synthetic Fibrin-Derived Bß15-42 Peptide Delays Thrombus Resolution in a Mouse Model.

[Figure: see text].

The Fibrin-Derived Peptide Bß15-42 (FX06) Ameliorates Vascular Leakage and Improves Survival and Neurocognitive Recovery: Implications From Two Animal Models of Cardiopulmonary Resuscitation.

OBJECTIVES: The fibrin-derived peptide Bß15-42 (FX06) has been proven to attenuate ischemia/reperfusion injury. We tested the hypothesis that Bß15-42 improves survival rate and neurocognitive recovery after cardiopulmonary resuscitation. DESIGN: Pig and mouse model of cardiopulmonary resuscitation. SETTING: Two university hospitals. SUBJECTS: Pigs and mice. INTERVENTIONS: Pigs (n = 16) were subjected to 8-minute cardiac arrest. Successful resuscitated pigs (n = 12) were randomized either to 3 mg/kg Bß15-42 followed by a continuous infusion of 1 mg/kg/hr for 5 hours (pFX06; n = 6) or the control group (pCONTROL; n = 6). Cardiac damage, function, and hemodynamics were recorded up to 8 hours. Mice (n = 52) were subjected to 4-minute cardiac arrest followed by cardiopulmonary resuscitation, and randomized either to two boli of 2.4 mg/kg Bß15-42 (mFX06; n = 26) or the control group (mCONTROL; n = 26). Fourteen-day survival rate, neurocognitive function, and endothelial integrity (additional experiment with n = 26 mice) were evaluated. MEASUREMENTS AND MAIN RESULTS: Bß15-42 reduced cumulative fluid intake (3,500 [2,600-4,200] vs 6,800 [5,700-7,400] mL; p = 0.004) within 8 hours in pigs. In mice, Bß15-42 improved 14-day survival rate (mFX06 vs mCONTROL; 11/26 vs 6/26; p < 0.05) and fastened neurocognitive recovery in the Water-Maze test (15/26 vs 9/26 mice with competence to perform test; p < 0.05). Bß15-42-treated mice showed a significant higher length of intact pulmonary endothelium and reduced pulmonary leukocyte infiltration. CONCLUSIONS: This study confirms the new concept of an important role of fibrin derivatives in global ischemia/reperfusion injury, which can be attenuated by the fibrin-derived peptide Bß15-42.

Effects of FX06 in vitro on platelet, coagulation, and fibrinolytic biomarkers in volunteers and patients with documented coronary artery disease.

FX06 is a naturally occurring fibrin-derived peptide demonstrated to confer cytoprotection in the setting of primary percutaneous coronary intervention. Because the effect of FX06 on human platelet, coagulation, and fibrinolysis biomarkers (PCFB) is unknown but is important for further clinical development, we evaluated how FX06 affects PCFB. The in vitro effects of the whole-blood pre-incubation with escalating concentrations of FX06 (4, 25, and 75 µg/mL) were assessed in aspirin-naïve healthy volunteers (n = 10), those with multiple risk factors for vascular disease (n = 10), and patients with documented coronary artery disease (n = 10). The last two groups were treated with aspirin (81 mg/daily). Thirty-two variables of PCFB were measured with the vehicle and for each chosen FX06 dose. Pretreatment of blood samples with FX06 resulted in a moderate but significant and mostly dose-dependent increases of platelet aggregation induced by adenosine diphosphate and collagen. Similarly, the closure time was reduced, suggesting share-induced activation, PECAM-1, GP Ib, GP IIb/IIIa activity, and vitronectin receptors, which were also up-regulated. In contrast, P-selectin and GPIIb antigen expression were reduced after FX06. All other PCFB were predominantly unaffected by FX06, with the exception of the increased plasminogen, decreased protein C activity, and activated von Willebrand factor. We conclude that in the therapeutic range, FX06 in vitro mildly affects hemostasis by way of mostly activating platelets. Applying moderate concomitant antiplatelet strategies should be considered for the adequate protection from vascular thrombotic events in patients treated with FX06. Similar ex vivo study in patients receiving aspirin and clopidogrel is warranted.

The NF-κB pathway: regulation of the instability of atherosclerotic plaques activated by Fg, Fb, and FDPs.

Recently, the molecular mechanism responsible for the instability of atherosclerotic plaques has gradually become a hot topic among researchers and clinicians. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play an important role in the processes of formation and development of atherosclerosis. In this study, we established and employed the transwell co-culture system of rabbit aortic endothelial cells and smooth muscle cells to explore the relationship between fibrin (Fb), fibrinogen (Fg), and/or their degradation products (FDPs) in relation to the instability of atherosclerotic plaques; meanwhile, we observed the effects of Fg, Fb, and FDPs on the mRNA levels of MMPs and VEGF as well as on the activation of nuclear factor-kappa B (NF-κB). We concluded that Fb, Fg, and FDPs are involved in the progression of the instability of atherosclerotic plaques via increasing the expression of MMPs and VEGF. This effect might be mediated by the NF-кB pathway.

A Retrospective Case-Control Study on the Diagnostic Values of Hemostatic Markers in Hypertensive Disorder of Pregnancy.

OBJECTIVE: The purpose of this study was to evaluate the diagnostic performance of the following hemostatic markers in hypertensive disorder of pregnancy (HDP): tissue-type plasminogen activator and inhibitor-1 complex (tPAI-C), thrombomodulin, thrombin-antithrombin complex, plasmin inhibitor-plasmin complex, D-dimer, and fibrinogen degradation products. METHODS: A total of 311 individuals diagnosed with HDP and 187 healthy controls (HC) of matched gestational age were admitted, including 175 subjects with gestational hypertension, 94 with mild preeclampsia, and 42 with severe preeclampsia. RESULTS: Compared with those of the HC group, the plasma concentrations of all the hemostatic markers continuously increased with the clinical severity of the hypertensive disorder, regardless of their statistical significance. In the receiver operating characteristic analysis, tPAI-C displayed the best discrimination performance. CONCLUSION: The tPAI-C level was consistently and significantly elevated across the different HDP groups when compared with the HC group, suggesting aggravated fibrinolysis disorder increasing with the severity of the HDP.

Pravastatin inhibits C-reactive protein generation induced by fibrinogen, fibrin and FDP in isolated rat vascular smooth muscle cells.

OBJECTIVE: The available evidence indicates that C-reactive protein (CRP) participates directly in atherosclerosis formation as an inflammatory molecule. Our previous investigation suggested that fibrinogen, fibrin and fibrinogen degradation products (FDP) produce a pro-inflammatory effect on vascular smooth muscle cells (VSMCs) through inducing CRP generation. In the present study, we observed the effect of pravastatin on CRP generation induced by fibrinogen, fibrin and FDP in rat VSMCs. METHODS: VSMCs from Sprague-Dawley rats were cultured. Fibrinogen, fibrin and FDP were used as stimulants for CRP generation. VSMCs were preincubated with pravastatin at 10, 30, 100 µM for 30 min prior to stimulation. CRP mRNA expression was studied by reverse transcription polymerase chain reaction (RT-PCR). CRP levels in the supernatant of VSMCs were measured by enzyme-linked immunosorbent assay (ELISA). CRP expression in VSMCs was examined with immunocytochemical staining. RESULTS: ELISA analysis showed that the pravastatin concentration-dependently reduced fibrinogen-, fibrin- and FDP-stimulated generation of CRP in VSMCs, with maximal inhibition of 56.6, 55.7 and 62.3%, respectively. Immunocytochemical staining and RT-PCR revealed that pravastatin inhibited protein and mRNA expression of CRP in VSMCs significantly. CONCLUSIONS: Pravastatin at the concentrations used in the present experiment has ability to relieve vascular inflammation and to restrain atherosclerotic processes via inhibiting the CRP production induced by fibrinogen, fibrin and FDP in VSMCs, which helps explain the beneficial effects of pravastatin on atherosclerosis.

The fibrin-derived peptide Bbeta15-42 protects the myocardium against ischemia-reperfusion injury.

In the event of a myocardial infarction, current interventions aim to reopen the occluded vessel to reduce myocardial damage and injury. Although reperfusion is essential for tissue salvage, it can cause further damage and the onset of inflammation. We show a novel anti-inflammatory effect of a fibrin-derived peptide, Bbeta15-42. This peptide competes with the fibrin fragment N-terminal disulfide knot-II (an analog of the fibrin E1 fragment) for binding to vascular endothelial (VE)-cadherin, thereby preventing transmigration of leukocytes across endothelial cell monolayers. In acute or chronic rat models of myocardial ischemia-reperfusion injury, Bbeta15-42 substantially reduces leukocyte infiltration, infarct size and subsequent scar formation. The pathogenic role of fibrinogen products is further confirmed in fibrinogen knockout mice, in which infarct size was substantially smaller than in wild-type animals. Our findings conclude that the interplay of fibrin fragments, leukocytes and VE-cadherin contribute to the pathogenesis of myocardial damage and reperfusion injury. The naturally occurring peptide Bbeta15-42 represents a potential candidate for reperfusion therapy in humans.

Prognosis analysis and risk factors related to progressive intracranial haemorrhage in patients with acute traumatic brain injury.

BACKGROUND: Since progressive intracranial haemorrhage (PIH) was introduced in neurosurgical literatures, several studies have been performed. PIH has been shown to be associated with a high increase in the risk of clinical worsening and related to morbidity and mortality as well. So, early detection and prediction of PIH is practically important in a clinical situation. OBJECTIVES: To investigate the risk factors related to PIH in patients with acute traumatic brain injury (TBI) and analyse their clinical significances. METHODS: PIH was confirmed by comparing the first and repeated CT scans. Data compared included gender, age, mechanism of injury, Glasgow Coma Score (GCS) at admission, timing from injury to the first CT, the signs of the initial CT scan, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fg), thrombin time (TT), platelet (PLT) and D-dimer (D-D) values. Logistic regression analysis was used to show the risk factors related to PIH. RESULTS: A cohort of 498 patients with TBI was evaluated, and there were 139 (27.91%) patients who suffered from PIH. The differences between PIHs and non-PIHs were significant in age, GCS at admission, the signs of the initial CT scan (fracture, subarachnoid haemorrhage, brain contusion and primary haematoma), PT, Fg and D-D values (p < 0.001). Logistic regression analysis was used to identify that CT scans (subarachnoid haemorrhage, brain contusion and primary haematoma) and plasma D-D values as the most important predictors of PIH (p < 0.001). CONCLUSIONS: For patients with the initial CT scan showing subarachnoid haemorrhage, brain contusion and primary haematoma with abnormal D-D levels, an earlier and dynamic CT scan should be performed, for the detection of PIH as early as possible and the medical intervention would be enforced in time.

Fibrinolysis is down-regulated in mouse collagen-induced arthritis, but its normalization does not alleviate the course of disease.

OBJECTIVE: Down-regulation of fibrinolysis and increased fibrin deposition in joints are hallmarks of rheumatoid arthritis (RA), and are believed to be involved in disease progression. The mouse model of collagen-induced arthritis (CIA) closely resembles RA and has been used to explore mechanism and treatments of RA, but neither the fibrinolytic system nor pro-fibrinolytic therapies were investigated in CIA. MATERIALS AND METHODS: Plasmin activity, levels of plasminogen activator inhibitor (PAI-1), D-dimer, and IL-6 were measured in plasma of CIA mice. Fibrin deposition and PAI-1 levels were also measured in inflamed joints. Mice were treated with plasminogen activators uPA (urokinase-type plasminogen activator) or tPA (tissue-type plasminogen activator). Effects of treatment on disease severity and fibrinolytic system were assessed. RESULTS: CIA caused decrease in plasmin activity, accompanied by increase in PAI-1 levels, in both blood and inflamed joints. This resulted in massive fibrin deposition in synovium. PAI-1 levels correlated negatively with plasmin activity and positively with IL-6. Treatments with uPA and tPA improved plasmin activity and removed fibrin deposits in inflamed joints. However, disease severity remained unchanged. CONCLUSIONS: Fibrinolytic changes in CIA parallel changes in RA, making CIA a suitable model to study fibrinolysis in RA. Normalization of plasmin activity in CIA after treatment with plasminogen activators had no effect on disease severity.

Integrin alpha(M)beta(2)-mediated cell migration to fibrinogen and its recognition peptides.

Leukocyte migration is the hallmark of inflammation, and integrin alpha(M)beta(2) and its ligand fibrinogen (Fg) are key participants in this cellular response. Cells expressing wild-type or mutant alpha(M)beta(2) and Fg or its derivatives have been used to dissect the molecular requirements for this receptor-ligand pair to mediate cell migration. The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 alpha(M)beta(2) recognition peptides support a chemotactic response; (b) when the I domain of alpha(L) was replaced with the I domain of alpha(M), the chimeric receptor supported cell migration to Fg; however, the alpha(M) subunit, containing the I domain but lacking the beta(2) subunit, supported migration poorly, thus, the alpha(M)I domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the beta(2) subunit and alpha(M)I domain; and (c) in addition to supporting cell migration, P2 enhanced alpha(M)beta(2)-mediated chemotaxis to Fg and the P1 peptide. This activation was associated with exposure of the activation-dependent epitope recognized by monoclonal antibody 7E3 and was observed also with human neutrophils. Taken together, these data define specific molecular requirements for alpha(M)beta(2) to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

Fibrinogen protects neutrophils from the cytotoxic effects of histones and delays neutrophil extracellular trap formation induced by ionomycin.

Neutrophils are pivotal players in immune defence which includes a process of release of histones and DNA as neutrophil extracellular traps (NETs). Histones, while toxic to invading pathogens, also kill host cells, including neutrophils. Bacteria have evolved mechanisms to escape neutrophils, including the secretion of leucocidins (e.g. ionomycin). Live cell video microscopy showed how fibrinogen and fibrin influence NETosis and neutrophil responses to extracellular histones. Histones were rapidly lethal to neutrophils after binding to cells, but formation of fibrinogen/fibrin-histone aggregates prevented cell death. Histone cytotoxicity was also reduced by citrullination by peptidyl arginine deiminase 4, or digestion by serine proteases. Ionomycin and phorbol 12-myristate 13 acetate (PMA) are used to trigger NETosis. Fibrinogen was responsible for a second distinct mechanism of neutrophil protection after treatment with ionomycin. Fibrinogen clustered on the surface of ionomycin-stimulated neutrophils to delay NETosis; and blocking the ß integrin receptor, αMß2, abolished fibrinogen protection. Fibrinogen did not bind to or protect neutrophils stimulated with PMA. Fibrinogen is an acute phase protein that will protect exposed cells from damaging circulating histones or leucocidins; but fibrinogen depletion/consumption, as in trauma or sepsis will reduce protection. It is necessary to consider the role of fibrinogen in NETosis.

Fibrinogen, fibrin, and FDP induce C-reactive protein generation in rat vascular smooth muscle cells: pro-inflammatory effect on atherosclerosis.

Atherosclerosis is a chronic inflammatory disease in the vessel. As an inflammatory cytokine, C-reactive protein (CRP) participates in the pathogenesis of atherosclerosis through multiple bioactivities. It has been widely accepted that hyperfibrinogenemia is associated with the formation and progression of atherosclerosis. But, it is unknown whether fibrinogen exerts a pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe the effect of fibrinogen, fibrin, and fibrin degradation products (FDP) on CRP generation in VSMCs. CRP mRNA expression was identified with the reverse transcription polymerase chain reaction. CRP level in the supernatant of VSMCs was measured with the enzyme-linked immunosorbent assay. CRP expression in VSMCs was examined with the immunocytochemical method. The results showed that fibrinogen, fibrin, and FDP all induced CRP production in VSMCs both in mRNA level and in protein level in a time- and concentration-dependent manner. The potency is FDP>fibrin>fibrinogen, which seems to mean that their pro-inflammatory activity decreases with increase of molecular weight of these three proteins. The finding provides a new mechanism for atherogenic effect of fibrin(ogen) and FDP, and emphasizes the importance of therapy of hyperfibrinogenemia in atherosclerosis.

Disorganization of cultured vascular endothelial cell monolayers by fibrinogen fragment D.

Fibrinogen fragment D, which is heterogeneous, has several important biological functions. Human fibrinogen fragments D94 (molecular weight, 94,000), D78 (78,000), and E (52,000) were purified. Fragments D78 and D94 but not purified fibrinogen or fragment E specifically caused disorganization of bovine aortic endothelial cells cultured as monolayers. Within 2 hours of exposure to pathophysiological concentrations of fragment D, the confluent endothelial cells retracted from each other and projected pseudopodia. These disturbed cells subsequently became rounded and detached from the substrate. The actin present in stress fibers in stationary monolayer cells was diffusely redistributed in cells with fragment D-induced alterations in morphology. This effect was not observed in monolayers of kidney epithelial cells. The results demonstrate a specific effect of fibrinogen fragment D on the disorganization of cultured vascular endothelial cell monolayers and suggest that fragment D plays a role in the pathogenesis of syndromes with vascular endothelial damage.

A double blind, single centre, sub-chronic reperfusion trial evaluating FX06 following haemorrhagic shock in pigs.

OBJECTIVE: Haemorrhagic shock causes ischaemia and subsequent fluid resuscitation causes reperfusion injury, jointly resulting in high morbidity and mortality. We tested whether the anti-inflammatory fibrin-derived peptide, Bbeta(15-42), also called FX06, is tissue protective in a model of haemorrhagic shock. METHODS: In a pig model, we standardised the severity of haemorrhagic shock by achieving a cumulative oxygen deficit of approximately 100ml/kg body weight by withdrawing blood over a period of 1h. This was followed by resuscitation with shed blood and full electrolyte solution, and pigs were monitored for 3 days. At reperfusion, 17 pigs were randomly assigned to FX06 or solvent treatment. RESULTS: FX06-treated pigs demonstrated improved cardiac function (stroke volume index: 67ml/m(2) versus 33ml/m(2)), decreased troponin T release in the early reperfusion (0.24ng/ml versus 0.78ng/ml), decreased AST levels after 24h (106U/l versus 189U/l) and decreased creatinine levels after 24h (108micromol/l versus 159micromol/l). Furthermore, FX06-treated pigs demonstrated preservation of the gut/blood barrier, while controls demonstrated high endotoxin plasma levels indicating translocation of bacteria and/or its products (0.2EU/ml versus 24.3EU/ml) after 24h. This study also demonstrates a significantly improved neurological performance in the FX06 group as determined by S100beta serum levels (0.72microg/l versus 1.25microg/l) after 48h and neurological deficit scores (11 versus 70) after 24h. CONCLUSION: FX06 - when administered as an adjunct to fluid resuscitation therapy - is organ protective in pigs. Further investigations are warranted to reveal the protective mechanism of FX06.

Binding to carboxypeptidase M mediates protective effects of fibrinopeptide Bß15-42.

During fibrinolysis a 28-amino-acid peptide is generated besides other degradation products of fibrin. This peptide, called Bß15-42, which is cleaved by plasmin from the end of the fibrin Bß-chain, is protective in myocardial and renal ischemia/reperfusion injury and improves the outcome in experimental sepsis. Bß15-42 has been shown to mediate different beneficial effects in endothelial cells through binding to vascular endothelial-cadherin. Here, we provide in vitro and in vivo evidence that Bß15-42 has additional cell protective activity in tubular cells, which is caused by a distinct mechanism. As vascular endothelial-cadherin is not expressed by tubular cells we used ligand-receptor capture technology LRC-TriCEPS to search for tubular cell surface receptors and identified carboxypeptidase M (CBPM) as a novel binding partner of Bß15-42. Silencing CBPM with siRNA reduced the protective potential of Bß15-42 against tubular cell stress. Bß15-42 inhibited the enzymatic activity of CBPM and modified the impact of CBPM on bradykinin signaling. We conclude that beneficial properties of Bß15-42 are not restricted to endothelial cells but are also active in epithelial cells where cytoprotection depends on CBPM binding.

Fibrin-VLDL Receptor-Dependent Pathway Promotes Leukocyte Transmigration by Inhibiting Src Kinase Fyn and is a Target for Fibrin ß15-42 Peptide.

According to the current view, binding of fibrin degradation product E1 fragment to endothelial VE-cadherin promotes transendothelial migration of leukocytes and thereby inflammation, and fibrin-derived ß15-42 peptide reduces leukocyte transmigration by competing with E1 for binding to VE-cadherin and, in addition, by signaling through Src kinase Fyn. However, the very low affinity of ß15-42 to VE-cadherin raised a question about its ability to inhibit E1-VE-cadherin interaction. Further, our previous study revealed that fibrin promotes leukocyte transmigration through the very-low-density lipoprotein (VLDL) receptor (VLDLR)-dependent pathway and suggested a possible link between the inhibitory properties of ß15-42 and this pathway. To test such a link and the proposed inhibitory mechanisms for ß15-42, we performed in vitro experiments using surface plasmon resonance, enzyme-linked immunosorbent assay, and leukocyte transendothelial migration assay, and in vivo studies with wild-type and VLDLR-deficient mice using mouse model of peritonitis. The experiments revealed that ß15-42 cannot inhibit E1-VE-cadherin interaction at the concentrations used in the previous in vivo studies leaving the proposed Fyn-dependent signaling mechanism as a viable explanation for the inhibitory effect of ß15-42. While testing this mechanism, we confirmed that Fyn plays a critical role in controlling fibrin-induced transendothelial migration of leukocytes and found that signaling through the VLDLR-dependent pathway results in inhibition of Fyn, thereby increasing leukocyte transmigration. Furthermore, our in vivo experiments revealed that ß15-42 inhibits this pathway, thereby preventing inhibition of Fyn and reducing leukocyte transmigration. Thus, this study clarifies the molecular mechanism underlying the VLDLR-dependent pathway of leukocyte transmigration and reveals that this pathway is a target for ß15-42.

Effect of fibrinogenolytic products D and E on fibrinogen and albumin synthesis in the rat.

Previous studies are in conflict over the effect of infusing mixed fibrinogen-fibrin degradation products on fibrinogen synthesis, as determined by changes in fibrinogen concentration or by incorporation of labeled amino acids into fibrinogen. We have injected purified homologous fragments D1 and E into rats and measured their fibrinogen and albumin synthetic rates by the [14C]carbonate technique, a method that provides quantitative estimates of hepatic secretory protein synthesis. Fibrinogen fractional synthetic rates were increased 2.5 times in animals injected with fragment D1, compared with saline-injected controls. No increase were observed in fragment E-injected animals. Neither fragment produced changes in albumin synthesis. Fragment D increased plasma fibrinogen concentration, but did not raise plasma haptoglobin levels. These results suggest that fragment D is a regulator of fibrinogen synthesis.

A comparative study of the promotion of tissue plasminogen activator and pro-urokinase-induced plasminogen activation by fragments D and E-2 of fibrin.

Plasmin generation by equimolar concentrations of tissue plasminogen activator (t-PA), pro-urokinase (pro-UK), and urokinase (UK), and a twofold higher concentration of a plasmin-resistant mutant rpro-UK (Ala-158-pro-UK) was measured on a microtiter plate reader. The promoting effects on this reaction of equimolar concentrations of fibrinogen, soluble fibrin (Desafib), CNBr fragment FCB-2 (an analogue of fragment D), or purified fragment E-2 were compared. Plasmin generation by t-PA was moderately promoted by fibrinogen, substantially promoted by Desafib and FCB-2, but not at all promoted by fragment E-2. By contrast, plasmin generation by pro-UK or by Ala-158-pro-UK was not promoted either by fibrinogen, Desafib, or FCB-2, but was significantly promoted by fragment E-2. Plasmin generation by UK was not significantly promoted by any of the fibrin(ogen) preparations. Treatment of fragment E-2 by carboxypeptidase-B (CPB), eliminated its promotion of pro-UK and Ala-158-pro-UK-induced plasmin generation. Pretreatment of FCB-2 with plasmin slightly potentiated its promotion of t-PA activity. This effect of plasmin pretreatment of FCB-2 was reversed by CPB treatment. Plasmin pretreatment of FCB-2 did not induce any promotion of activity in pro-UK or Ala-158-pro-UK. The findings show that the intrinsic activity of pro-UK and the activity of t-PA are promoted by different regions of the fibrin(ogen) molecule. The latter is stimulated primarily by a determinant in the fragment D region, which is available in intact fibrin. By contrast, plasminogen activation by the intrinsic activity of pro-UK was stimulated exclusively by fragment E-2, which is unavailable in intact fibrin. The findings are believed relevant to fibrinolysis and support the concept that t-PA and pro-UK are complementary, sequential, and synergistic in their actions.

Degradation products of fibrinogen by elastase-like neutral protease from human granulocytes. Characterization and effects on blood coagulation in vitro.

We investigated the effect of elastase-like neutral protease isolated from human granolocytes on human fibrinogen. Dependent on enzyme concentration and time of incubation, the elastase-like protease induced a progressive degradation of fibrinogen. Analysis of the remaining polypeptide chains showed a high susceptibility of the Aalpha- and low susceptibility of the gamma-chain of fibrinogen towards the proteolytic action of the enzyme. The split products were characterized by polyacrylamide gel electrophoresis and two-dimensional immunoelectrophoresis. They showed antigenic determinants of fibrinogen and of plasmin-induced proteolysis products D and E. The cleavage fragments isolated by gel chromatography had distinct molecular weights. Coagulability of fibrinogen by thrombin was inhibited according to the concentration of the protease and the time of incubation. Split products of fibrinogen with higher molecular weight prolonged the coagulation time of native fibrinogen, whereas low molecular weight fragments were ineffective.