Fibrin generation and digestion in patients with angina pectoris

Summary Fibrin generation and lysis were studied in 28 patients with angina pectoris (14 with active disease and 14 with inactive disease) and in 14 normal controls. The fibrinolytic response was evaluated by comparing the ratio between the plasma levels of fibrinopeptide A and fibrin degradation products. Levels of both were higher in patients than in controls (P<0.001), with higher levels in active than in inactive disease (P<0.001). The fibrinopeptide A/fibrin degradation products ratio was much higher (P<0.001) in the active group than in other groups. Thus, in patients with angina pectoris, especially in the active state, the increased thrombin generation is not paralleled by an equivalent increase in fibrinolytic activity.     

Effect of low-dose heparin on fibrinogen levels in patients with chronic ischemic heart disease

Several prospective studies have demonstrated that high plasma fibrinogen levels are associated with an increased risk of ischemic heart disease. Since in most patients an increased thrombin generation has been reported, we investigated whether the control of thrombin generation could affect plasma fibrinogen levels. Forty male outpatients (20 asymptomatic with previous myocardial infarction and 20 with stable effort angina) were enrolled in a randomized medium-term (6 months) cross-over study. Clottable fibrinogen, according to Clauss, prothrombin fragment 1+2, thrombin-antithrombin complex, and fibrinopeptide A were evaluated in relation to treatment with low-dose heparin. After a 15-day wash-out period, during which patients had been treated only with nitrates if needed, patients were allocated to two sequential periods of treatment with standard heparin (12,500 U, subcutaneously daily) plus antianginal treatment or antianginal treatment alone, separated by a second 15-day wash-out period. At the end of the treatment period with low-dose heparin significant decreases in the plasma fibrinogen (2.5±0.6 g/l vs. 3.3±0.5 g/l,P<0.001), prothrombin fragment 1+2 (1.4±0.5 nmol/l vs. 1.9±0.7 nmol/l,P<0.001), thrombinantithrombin (4.5±2.4 ng/ml vs. 9.7±3.6 ng/ml,P<0.001), and fibrinopeptide A (2.1±1.1 ng/ml vs. 3.5±2.1 ng/ml,P<0.001) were observed compared with the period without heparin. The present results indicate that low-dose heparin can effectively control the increased abnormal thrombin generation and elevated fibrinogen levels in patients with ischemic heart disease, possibly decreasing the risk of cardiovascular death.     

D-Dimer plasma levels during normal pregnancy measured by specific ELISA

A progressive increase in D-dimer plasma levels together with an increase in fibrinogen has been previously reported during normal pregnancy. However, significantly different D-dimer levels have been observed in different assays, due to different specificity of the antibodies employed. The aim of this study was to verify the increase in fibrin degradation product levels during normal pregnancy, using a recently introduced specific D-dimer ELISA. We determined D-dimer (ELISA) and fibrinogen (clotting method) plasma levels in 63 normal pregnant women, during three different periods of pregnancy (A, 7–20 weeks; B, 21–30 weeks; C, > 30 weeks). During period A, D-dimer plasma levels (range 2–103 ng/ml) showed an insignificant increase compared with a control group of non-pregnant women (range 2–73 ng/ml). During periods B and C, we observed an increase in D-dimer level (P<0.0001) compared with period A, with a significant correlation between D-dimer levels and gestational age (P<0.0001). Period A fibrinogen levels (range 3.24–6.43 g/l) were significantly higher (P<0.0001) than in controls (range 2.31–4.71 g/l), with a further increase in periods B and C. In conclusion, we confirmed a progressive increase in plasma concentrations of fibrin degradation product during normal pregnancy, but D-dimer levels were significantly lower than those reported in the literature for other ELISAs.     

Thrombus formation and platelet-vessel wall interaction in the nephrotic syndrome under flow conditions.

Increased in vitro platelet aggregability and hypercoagulability are generally held to be main determinants in the prethrombotic state in nephrosis. In vivo, however, thrombotic events depend on the dynamic interaction of flowing blood with the vessel wall. The present study confirms that aggregability of platelets of nephrotic patients is significantly increased by mere stirring or by exogenous stimuli as adenosine diphosphate and arachidonic acid. Moreover, the nephrotic patients have high von Willebrand factor and decreased red blood cell deformability, which normally increase platelet-vessel wall interaction. However, perfusion studies under well-defined flow conditions, in which anticoagulated nephrotic blood was exposed to deendothelialized human umbilical artery segments and sprayed collagen, showed normal platelet adhesion and only a modest increase in the deposition of platelet aggregates. This suggests that some factor counteracts platelet-vessel wall interaction under flow conditions in the nephrotic syndrome. When tissue factor associated with endothelial extracellular matrix (ECM) was allowed to generate thrombin, perfusions with nephrotic blood over this ECM resulted in a strong increase in fibrin generation. The capacity of patient blood to form increased amounts of fibrin appeared strongly correlated with the level of hyperfibrinogenemia. Platelet adhesion as well as aggregation in these experiments was even decreased below control values. This suggests that fibrin coverage may block the direct contact between blood platelets and matrix. We therefore also studied the isolated effect of high fibrinogen on platelet-vessel wall interaction by increasing fibrinogen concentrations in normal blood. Modulation of fibrinogen concentrations in normal blood could mimic all the observations in nephrotic blood: platelet aggregation in suspension increased with increasing concentrations of fibrinogen, while platelet adhesion and aggregate formation under flow conditions decreased. In perfusions over tissue factor-rich matrix, fibrin deposition increased. Therefore, our observations indicate that nephrotic hyperaggregability in suspension is not associated with increased platelet vessel wall-interaction under flow conditions. The latter is probably counteracted by high levels of fibrinogen. Our observations further suggest that hyperfibrinogenemia may be a major thrombotic risk factor in nephrosis by inducing more fibrin depositions.     

Membrane fatty acids and erythrocyte Li-Na countertransport in nephrotic syndrome and their relationship

Summary Cholesterol/phospholipids molar ratio and fatty acid composition have been estimated in erythrocyte membrane of 12 patients suffering from nephrotic syndrome and compared to values obtained in 23 normal subjects matched for sex and age. The membrane lipid composition has been correlated with the activity of erythrocyte Li-Na countertransport of the same subjects. The results show a significant increase in cholesterol/phospholipids ratio and total saturated fatty acids when erythrocytes of nephrotic patients are compared to normal erythrocytes, whereas total unsaturated fatty acids were lower in nephrotics (p<0.002). Li-Na countertransport was higher in nephrotics (p<0.001) and it was positively correlated with the total amount of saturated fatty acids of the erythrocyte membrane (r=+0.451; p<0.01). On the contrary, Li-Na countertransport was negatively correlated with the total amount of unsaturated fatty acids (r=−0.468; p<0.01). This work was supported by a grant (84.857.04) fromConsiglio Nazionale delle Ricerche (CNR), Roma, Italy.     

SIGNIFICANCE OF CRYOPROFIBRIN IN FIBRINOGEN-FIBRIN CONVERSION

Fibrinogen altered by thrombin-catalyzed liberation of fibrinopeptide A was found to combine with native fibrinogen to form a cold-precipitable complex we have called "cryoprofibrin." The altered fibrinogen lacking fibrino-peptide A polymerized into fibrin, but not until conditions for equilibrium between its incorporation into both cryoprofibrin and fibrin were satisfied. At equilibrium, the concentration of cryoprofibrin was maintained at a threshold proportional to the concentration of fibrinogen. When the concentration of cryoprofibrin was below threshold, fibrin could be depolymerized and solubilized by fibrinogen with resultant formation of cryoprofibrin. Since threshold concentrations of cryoprofibrin appear necessary for precipitation of fibrin, the concentration of cryoprofibrin in plasma provides a basis for determining intravascular deposition of fibrin. Intravascular deposition of fibrin does not appear to occur normally in rabbits, because the concentration of cryoprofibrin in plasma from normal rabbits is far below the threshold for precipitation of fibrin. The applicability of cryoprofibrin as an indicator of fibrin deposition is demonstrated by the occurrence of levels of cryoprofibrin approaching the threshold for precipitation of fibrin in plasma from endotoxin-treated rabbits. The current concept that the fibrinogen molecule can dissociate into subunits can be used to explain the conversion of fibrinogen to cryoprofibrin. As one possibility, the two residues of fibrinopeptide A contained in fibrinogen may be located on two separate subunits of the molecule; cryoprofibrin is produced when one of these subunits is replaced by a subunit altered by loss of fibrinopeptide A. Recombination of native subunits with subunits altered by loss of A would counter dissociation of cryoprofibrin and inhibit polymerization of subunits lacking fibrinopeptide A. As an alternate mechanism, two residues of A may be liberated concurrently from a single subunit. Cryoprofibrin would then correspond to a fibrinogen molecule, containing a subunit with two residues of A, in combination with an altered molecule containing a subunit lacking two residues of A. Liberation of fibrinopeptide B did not contribute measurably to production of fibrin resulting from limited action of thrombin on rabbit fibrinogen. Both fibrin containing B but not A, and fibrin containing neither B nor A, as is produced by extensive action of thrombin, could be solubilized by fibrinogen. Thrombin, or another enzyme utilizing tosyl-L-arginine methyl ester as substrate, appeared reversibly to inhibit polymerization of fibrin containing fibrinopeptide B. This enzyme and fibrinogen were the only proteins appearing to inhibit polymerization in plasma from normal rabbits.     

Cellular basis for blunted volume expansion natriuresis in experimental nephrotic syndrome.

Experimental nephrotic syndrome results in sodium retention, reflecting, at least in part, an intrinsic defect in renal sodium handling in the distal nephron. We studied the relationships among plasma atrial natriuretic peptide (ANP) concentration, sodium excretion (UNaV), and urinary cyclic GMP excretion (UcGMPV) in vivo, and the responsiveness of isolated glomeruli and inner medullary collecting duct (IMCD) cells to ANP in vitro, in rats with adriamycin nephrosis (6-7 mg/kg body weight, intravenously). 3-5 wk after injection, rats were proteinuric and had a blunted natriuretic response to intravenous infusion of isotonic saline, 2% body weight given over 5 min. 30 min after onset of the infusion, plasma ANP concentrations were elevated in normals and were even higher in nephrotics. Despite this, nephrotic animals had a reduced rate of UcGMPV after the saline infusion, and accumulation of cGMP by isolated glomeruli and IMCD cells from nephrotic rats after incubation with ANP was significantly reduced compared to normals. This difference was not related to differences in binding of 125I-ANP to IMCD cells, but was abolished when cGMP accumulation was measured in the presence of 10(-3) M isobutylmethylxanthine or zaprinast (M&B 22,948), two different inhibitors of cyclic nucleotide phosphodiesterases (PDEs). Infusion of zaprinast (10 micrograms/min) into one renal artery of nephrotic rats normalized both the natriuretic response to volume expansion and the increase in UcGMPV from the infused, but not the contralateral, kidney. These results show that, in adriamycin nephrosis, blunted volume expansion natriuresis is associated with renal resistance to ANP, demonstrated both in vivo and in target tissues in vitro. The resistance does not appear related to a defect in binding of the peptide, but is blocked by PDE inhibitors, suggesting that enhanced cGMP-PDE activity may account for resistance to the natriuretic actions of ANP observed in vivo. This defect may represent the intrinsic sodium transport abnormality linked to sodium retention in nephrotic syndrome.     

Evidence that factor VII levels correlate strongly with fibrinopeptide A release: evaluation by an ex vivo method

Summary The generation of thrombin was estimated by an assay for fibrinopeptide A which was developed and employed to evaluate the relationship between factor VII and thrombin release. The amount of fibrinopeptide A released correlated strongly, when assayed in the early stages of the reaction, with factor VII coagulant activity levels in the rage 50–2,000 units/dl. The method was then applied to study the relationship between factor VII and fibrinopeptide A in plasma from blood donors, women on the pill and patients on oral anticoagulants (with a range of factor VII coagulant activity from 8.5 to 600 units/dl). The overall evaluation of the relationship between factor VII and fibrinopeptide A showed a strong correlation which was higher for factor VII coagulant activity (r=0.90) than for factor VII antigen levels (r=0.817). The regression analysis which best fitted the data was the multiplicative one, indicating that thrombin formation increases faster when factor VII coagulant activity is in the upper part of the normal range or higher. In patients on oral anticoagulants, the correlation between factor VII and fibrinopeptide A was very poor. Our data fit well with the findings of the epidemiological studies in which high levels of factor VII coagulant activity were shown to be associated with an increased incidence of data coronary artery disease.     

Plasma soluble fibrin monomer complexes in nephrotic syndrome--with reference to hypoalbuminemia

Plasma soluble fibrin monomer complexes (SFMC) in 10 patients with nephrotic syndrome were measured to demonstrate the contribution of hypoalbuminemia to the SFMC formation. The levels of SFMC as well as plasma fibrinogen (Fbg) levels were significantly higher than those in control subjects. There was a negative correlation between the levels of SFMC and serum albumin, and also between Fbg and serum albumin. The increase in SFMC levels which indicates the intravascular generation of thrombin might be correlated to hypoalbuminemia in nephrotic syndrome with hypercoagulability.     

Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of Dacron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrin-rich venous-type thrombus. Thrombosis was quantified by 111In-platelet imaging and 125I-fibrinogen accumulation. Intravenous infusion of alpha-thrombin, 1-2 U/kg-min for 1 h, increased baseline activated PC levels (approximately 5 ng/ml) to 250-500 ng/ml (P < 0.01). The lower thrombin dose, which did not deplete circulating platelets, fibrinogen, or PC, reduced arterial graft platelet deposition by 48% (P < 0.05), and platelet and fibrin incorporation into venous-type thrombus by > 85% (P < 0.01). Thrombin infusion prolonged the activated partial thromboplastin clotting time, elevated fibrinopeptide A (FPA), thrombin-antithrombin III complex (T:AT III), and fibrin D-dimer plasma levels (P < 0.01), but did not affect bleeding times. Thrombin's antithrombotic effects were blocked by infusing a monoclonal antibody (HPC-4) which prevented PC activation in vivo, caused shunt occlusion, increased the consumption of platelets and fibrinogen, elevated plasma FPA and T:AT III levels, and reduced factor VIII (but not factor V) procoagulant activity (P < 0.05). We conclude that activated PC is a physiologic inhibitor of thrombosis, and that activation of endogenous PC may represent a novel and effective antithrombotic strategy.     

Fibrinopeptide A cleavage and platelet release in whole blood in vitro. Effects of stimuli, inhibitors, and agitation.

The relationship between platelet release and fibrinopeptide A cleavage from fibrinogen to form fibrin I in vitro was examined in blood allowed to clot undisturbed or with gentle agitation. In undisturbed or agitated blood platelet release and fibrin I formation occurred simultaneously. When hirudin was added to undisturbed blood it prevented platelet release as well as fibrin I formation. In contrast, hirudin added to agitated blood had little effect on platelet release despite complete inhibition of fibrin I formation. Collagen added to either undisturbed or agitated blood increased platelet release and then fibrin I formation, and ADP added to undisturbed blood caused an initial burst of platelet release followed by slight acceleration of fibrinopeptide A cleavage. Prostaglandin E1 and theophylline prevented platelet release in both undisturbed and agitated blood, but did not affect fibrin I formation. The results with inhibitors in agitated blood suggest that fibrin I formation and platelet release can occur independently in the presence of the increased interactions induced by agitation. Addition of thrombin or tissue thromboplastin to undisturbed blood accelerated fibrin I formation with little effect on platelet release. Finally, initial thrombin formation in undisturbed blood appeared to be associated with the platelet surface. These relationships suggest that thrombin formation via the intrinsic system leads to thrombin generation on the platelet surface and simultaneous platelet release and fibrin I formation, while thrombin generated via tissue thromboplastin leads to thrombin formation in the plasma and fibrin I formation preceding platelet release. Activation by interaction of blood with collagen causes initial acceleration of platelet release and later acceleration of fibrin I formation.     

Thrombin generation during reperfusion after coronary artery bypass surgery associates with postoperative myocardial damage

BACKGROUND: Cardiopulmonary bypass and coronary artery bypass grafting (CABG) result in significant thrombin generation and activation of fibrinolysis. Thrombin contributes to myocardial ischemia-reperfusion injury in animal studies, but the role of thrombin in myocardial damage after CABG is unknown. OBJECTIVES: We measured thrombin generation and fibrin turnover during reperfusion after CABG to evaluate their associations with postoperative hemodynamic changes and myocardial damage. METHODS: One hundred patients undergoing primary, elective, on-pump CABG were prospectively enrolled. Plasma prothrombin fragment F(1+2) and D-dimer were measured preoperatively and at seven time points thereafter. Mass of the Mb fraction of creatine kinase (Ck-Mbm) and troponin T (TnT) were measured on the first postoperative day. RESULTS: Reperfusion induced an escalation of thrombin generation and fibrin turnover despite full heparinization. F(1+2) during early reperfusion associated with postoperative pulmonary vascular resistance index. F(1+2) at 6 h after protamine administration correlated with Ck-Mbm (r = 0.40, P < 0.001) and TnT (r = 0.44, P < 0.001) at 18 h postoperatively. Patients with evidence of myocardial damage (highest quintiles of plasma Ck-Mbm and TnT) had significantly higher F(1+2) during reperfusion than others (P < 0.002). Logistic regression models identified F(1+2) during reperfusion to independently associate with postoperative myocardial damage (odds ratios 2.5-4.4, 95% confidence intervals 1.04-15.7). CONCLUSIONS: Reperfusion caused a burst in thrombin generation and fibrin turnover despite generous heparinization. Thrombin generation during reperfusion after CABG associated with pulmonary vascular resistance and postoperative myocardial damage.     

Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma

Three recent studies have reported that fibrin in solution significantly inhibits the ability of heparin to catalyze the inhibition of thrombin by antithrombin III. In addition, heparin inhibits the release of fibrinopeptide A by clot-bound thrombin less effectively than it inhibits the release of fibrinopeptide A by thrombin in solution. We have also reported that dermatan sulfate, which catalyzes thrombin inhibition by heparin cofactor II, inhibits thrombus growth in rabbits more effectively than heparin. Because the results of these studies suggest that fibrin inhibits the reactivity of thrombin with antithrombin III-heparin but not with heparin cofactor II-dermatan sulfate, we compared the relative catalytic effects of heparin and dermatan sulfate on thrombin inhibition in plasma, both in the presence and absence of fibrin. We quantitated the rates of thrombin inhibition by antithrombin III and heparin cofactor II by specific enzyme-linked immunosorbent assays. When it was generated, fibrin was kept in solution by adding 2 mmol/L Gly-Pro-Arg-Pro to plasma. Fibrinogen-fibrin reduced the reactivity of thrombin with plasma antithrombin III, both in the presence of and in the absence of heparin. In contrast, the catalytic action of dermatan sulfate on thrombin inhibition by plasma heparin cofactor II was unimpaired by fibrinogen-fibrin. Based on the ability of dermatan sulfate to inhibit thrombus growth in rabbits, failure of fibrinogen-fibrin to moderate the catalytic action of dermatan sulfate may account for its greater antithrombotic effectiveness relative to that of heparin.     

Fibrin polymerization is crucial for thrombin generation in platelet-rich plasma in a VWF–GPIb-dependent process, defective in Bernard–Soulier syndrome

Summary.  Defective prothrombin consumption has been reported in the proband case of Bernard–Soulier syndrome (BSS). There is no consensus, however, on whether the formation of platelet procoagulant activity (PPA) is impaired in BSS and, if so, whether this is due to the lack of GPIb-V-IX-dependent binding of thrombin or of von Willebrand factor (VWF). We show thrombin generation (TG) in platelet-rich plasma of BSS (BSS-PRP) to be defective provided that fibrin remains present in the reaction mixture and that the giant platelets are not damaged by frequent subsampling. In BSS-PRP addition of (thrombin-free) fibrin did not increase TG as in normal PRP, supporting our previous hypothesis that the interaction of fibrin, VWF and GPIb triggers PPA development. Fibrin formed during the lag phase of TG by a snake venom enzyme which only removed fibrinopeptide A induced an immediate burst of TG, that was inhibited by a monoclonal antibody against GPIb (6D1) that abolishes ristocetin-induced binding of VWF to platelets. Inversely, inhibition of polymerization decreased TG and the residual activity was insensitive to 6D1. We conclude that polymerizing fibrin interacts with VWF so as to activate GPIb.     

Fibrinogen logroño. A new case of congenital dysfibrinogenemia

Summary An abnormal fibrinogen was discovered in a 9-year-old male subject without history of hemorrhagic diathesis. Coagulation time, prothrombin time and reptilase time were prolonged. The thrombin time was corrected using increasing concentrations of normal plasma and bovine thrombin; there was 2 partial correction at pH 6.5 and ionic strength 0.05. A study of the family showed that the mother and a brother of the propositus presented the same abnormalities. Analysis of the purified fibrinogen showed normal fibrinopeptide release and normal levels of sialic acid and hexosamines. However, coagulation index, polymerization of fibrin monomers, isoelectric point and sedimentation coefficient were abnormal. In view of the abnormalities described and by comparison with the data reported in the literature, we believe that this should be considered a new variant of the fibrinogen molecule and we have designated it ‘fibrinogen Logro?o’.     

Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.

TNF-alpha induces changes in endothelial cell functions, such as upregulation of tissue factor, resulting in endothelial procoagulant activity which may play a role in disseminated intravascular coagulation. The procoagulant activity of TNF-alpha-stimulated endothelial cell monolayers was studied in a human ex vivo native (nonanticoagulated) blood flow system using the three thrombin inhibitors recombinant hirudin, Ro 46-6240, and heparin. Under venous blood flow conditions (shear rate 65 s-1) recombinant hirudin, Ro 46-6240, and heparin inhibited fibrin deposition on the endothelial cells by 50% at concentrations of 14, 28, and 412 ng/ml, respectively. The highest tested concentrations of the thrombin inhibitors reduced the postchamber fibrinopeptide A levels from 713 +/- 69 to < 70 ng/ml. Surprisingly, even at relatively high inhibitor concentrations, some local fibrin deposits were found on TNF-alpha-stimulated cells, suggesting that some endothelial cells possess higher procoagulant activity than others. Therefore, the surface expression pattern of tissue factor, the primary initiator of coagulation in this system, was examined by immunogold-silver staining. The results showed that the tissue factor density on the cell surface varied strongly among TNF-alpha-stimulated endothelial cells. Using TNF receptor-selective agonistic mutants of TNF-alpha, it was demonstrated further that the heterogenous surface expression of tissue factor was mediated entirely by the 55-kD TNF receptor and did not involve the 75-kD TNF receptor. We conclude that in this system TNF-alpha induces heterogenous tissue factor expression which may lead to a high local thrombin concentration, such that even in the presence of thrombin inhibitors focal fibrin deposition occurs.     

Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors.

Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release mediated by fluid-phase thrombin with their activity against the clot-bound enzyme. Incubation of thrombin with citrated plasma results in concentration-dependent FPA generation, which reaches a plateau within minutes. In contrast, there is progressive FPA generation when fibrin clots are incubated with citrated plasma. Heparin, hirudin, hirudin dodecapeptide (hirugen), and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) produce concentration-dependent inhibition of FPA release mediated by fluid-phase thrombin. However, heparin is much less effective at inhibiting thrombin bound to fibrin because a 20-fold higher concentration is necessary to block 70% of the activity of the clot-bound enzyme than is required for equivalent inhibition of fluid-phase thrombin (2.0 and 0.1 U/ml, respectively). In contrast, hirugen and PPACK are equally effective inhibitors of fluid- and solid-phase thrombin, while hirudin is only 50% as effective against the clot-bound enzyme. None of the inhibitors displace bound 125I-labeled thrombin from the clot. These studies indicate that (a) clot-bound thrombin is relatively protected from inhibition by heparin, possibly because the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin, and (b) clot-bound thrombin is susceptible to inactivation by antithrombin III-independent inhibitors because the sites of their interaction are not masked by thrombin binding to fibrin. For these reasons, antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings.     

Thrombin and Plasmin Activity in Diabetes Mellitus and their Association with Glycaemic Control

Abnormalities of haemostasis are common in diabetes mellitus.As indicators of fibrinolysis and coagulation, plasmin and thrombinactivity were assessed by assay of the fibrinogen peptide derivativesBß_15-42 and fibrinopeptide A respectively in 60 diabeticpatients and 50 control subjects in a cross-sectional study.Glycosylated haemoglobin (HbA₁) correlated with Bß_15-42(r=0.26, p<0.05) and fibrinopeptide A (r=0.30, p<0.05)in the diabetic patients suggesting that poor glycaemic control(i.e high HbA₁ levels) was associated with depressed plasminand enhanced thrombin activity. Compared to controls, fibrinopeptideA levels were increased in diabetics (p<) irrespective ofsex or type of diabetes. Bß_15-42 levels were normalin diabetic females but increased in diabetic men (p<0.001)possibly secondary to the activation of coagulation. These resultssuggest that in diabetes mellitus activation of coagulationis the dominant haemostatic abnormality and that better glycaemiccontrol could influence in-vivo plasmin and thrombin activityfavourably.     

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Adhesion and spreading of cultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin beta chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B beta chain. After clotting with thrombin, this fibrin derivative lacking B beta 1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 15-42 of the fibrin beta chain.