Owen, Whyte G.

Association of heparin non-covalently with bovine factor Xa was analyzed by Superose-12 gel chromatography. In 0.05 M NaCl, 0.02 M Tris, pH 7.5, DEGR-Xa (factor Xa inactivated by dans-Glu-Gly-Arg-CH2Cl) was eluted as a single, sharp peak at Ve/Vt=0-65 (elution volume/internal volume). Mixtures of heparin and DEGR-Xa were eluted as two partially resolved peaks of protein at Ve/Vt=0.59 and 0.65. The fraction of DEGFUXa in the leading peak was directly proportional to [heparin], and at 100 yM heparin the leading peak contained more than half the total protein. When 0.02 M HEPES was substituted for Tris a single, slightly broadened peak at Ve/Vt=0.64 was obtained on chromatography of 100 μM heparin and 10 μM DEGR-Xa. In a buffer system comprising 0.02 M Tris, 0.02 M HEPES, 0.03 M NaCl, pH 7.5, two peaks were eluted at Ve/Vt=0.59 and 0.65. Therefore, Tris increases the affinity of DEGR-Xa for heparin.Solutions buffered with Tris or HEPES were compared for effects on the kinetics of inhibition of factor Xa by antithrombin III-heparin. Reaction mixtures containing 1 nM factor Xa, 30 nM heparin and 600 nM antithrombin III were assayed with S-2222 at intervals of 2-10 sec. Reagent concentrations were chosen (a) to assure pseudo-first-order kinetics, (b) to have [heparin]<< Kq for factor Xa-heparin, and (c) to bind virtually all available heparin to antithrombin III. The same second-order rate constant, Kobs=2.5×107 M−1s−1, was obtained in both buffer systems. We conclude that the association of factor Xa with heparin observed directly by gel chromatography does not contribute to the reaction rate of factor Xa with antithrombin III-heparin.

SummaryReversibility in vivo of acute platelet thrombosis in response to specific anticoagulants is analyzed with thrombi that develop in segments (1 cm) of porcine carotid arteries externally crushed with a hemostat. Most thrombi fill the lumens of the injured segments (ca. 1 cm × 3 mm, 1 × w) within 30 min and comprise masses of platelets interpenetrated with neutrophil-lined seepage channels of blood. Continuous quantitative assay of thrombus mass is provided by a gamma detector placed over the injured segments to collect counts from 111In-labeled platelets. Thrombi established 30 min after injury, otherwise stable for 6 h, clear during 30-60 min of continuous infusion of either hirudin, tick anticoagulant or activated porcine protein C, or intermittent activation of endogenous protein C with a latent thrombin reagent. Anticoagulant dose-dependence of thrombus clearance is established for hirudin between 0.01 and 1.0 mg/kg/min. Thrombi become progressively refractory to hirudin between 0.5 and 6 h after injury. Neither heparin nor low-molecular-weight heparin in full (clinical) anticoagulant doses yield significant dethrombosis. It is concluded that, within time limits, controlled thrombin generation in platelet thrombi maintains platelet cohesion without catalyzing irreversible platelet aggregation or clotting of fibrinogen.

SummaryThe relative contribution of plasmin, miniplasmin, PMN-elastase and cathepsin G to the fibrin-gel dissolution is studied. The global kcat/KM ratios are determined as a measure of the fibrinolytic catalytic efficiency using spectrophotometric kinetic analysis of the competition between fibrin and synthetic peptide substrates for the proteases, turbi-dimetric assay for fibrin dissolution and gel-filtration of the partially degraded fibrin. When the substrate is fibrin polymerized in the presence of 3 mM Ca2+, the value of this ratio is 4.3 × 105 M-1·s-1 for plasmin, 1.9 × 105 M-1·s-1 for miniplasmin, 5.0 × 104 M-1·s-1 for PMN-elastase and 2.2 × 103 M-1·s-1 for cathepsin G. When fibrin is polymerized without addition of Ca2+, the kcat/KM values are increased by a factor of 2.3 for plasmin, 2.0 for miniplasmin and 1.6 for cathepsin G, whereas that of PMN-elastase is unchanged. Progressive crosslinking of fibrin decreases the catalytic action of all studied proteases, but no change in their relative contribution to fibrinolysis is observed. When plasmin inhibitor (at physiological concentration) is also crosslinked to fibrin, the most efficient fibrinolytic enzymes are miniplasmin and PMN-elastase. The effect of 6-aminohexanoate on the formation of fibrin degradation products by plasmin and miniplasmin suggests that the high-affinity lysine binding site in the N-terminal kringle domain of plasmin is involved in the interactions with the native polymerized fibrin, whereas the fifth kringle found in both enzymes participates in binding to newly exposed lysine residues. These results provide a quantitative basis for the evaluation of fibrinolytic efficiency and support the concept of synergistic fibrinolysis.

SummaryThe plasmin substrate, H-D-norleucyl-hexahydrotyrosyl lysine-p-nitroanilide (Spectrozyme-PL), was found to be equiva lent to 6-aminohexanoate as an enhancer of porcine and human plasminogen activation by urokinase and of removal of the 1-77 peptide of plasminogen by plasmin. Activation of plasminogen lacking kringles 1-4, on the other hand, was not influenced by Spectrozyme PL. Although the rate of activation of human plasminogen and the modification of human plasminogen by plasmin are faster by an order of magnitude than that of the activation and modification of porcine plasminogen, both reactions in the human zymogen, the hydrolysis at arg561-val562 and at lys77lys78, are accelerated by Spectrozyme PL. The findings indicate that kinetic interpretation of plasminogen activation in solutions containing substrates, where the substrate has been incorporated to inhibit feedback proteolysis by plasmin, must account for the cofactor activity as well as the inhibitory activity of the substrate.

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.

Experiments were designed to determine how ovariectomy modulates mitogenic factors in platelets and how these factors affect proliferation of coronary arterial smooth muscle. Platelet-derived growth factors (PDGFABand PDGFBB), transforming growth factors (TGF-β1and TGF-β2), and vascular endothelial growth factor (VEGF165) were quantified in platelet lysates and platelet-poor plasma from adult gonadally intact and ovariectomized female pigs by ELISA. Proliferation of cultured coronary arterial smooth muscle cells (SMCs) from both groups of pigs was determined in response to autologous or heterologous platelet lysates. Platelet concentrations of PDGFBB, but not PDGFAB, TGF-β1, and TGF-β2, increased with ovariectomy. VEGF165was not detected in platelets from either group. Proliferation of SMCs from ovariectomized females was significantly greater on exposure to autologous or heterologous platelet lysates than proliferation of SMCs from intact females. These results indicate that ovariectomy increases concentrations of PDGFBBin platelets. Higher levels of PDGFBBin platelets in synergy with other platelet-derived products could contribute to increased proliferative arterial response to injury after ovariectomy.

BACKGROUND The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. METHODS AND RESULTS Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline). CONCLUSIONS Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.

We studied the ability of a new instrument, the PlaCor PRT that measures shear-induced platelet aggregation in fingerstick, non-anticoagulated blood without added agonists, to detect platelet dysfunction ex vivo. Platelet reactivity time (PRT) and whole blood aggregation (WBA) were measured in 160 healthy volunteers, before and after aspirin and in 170 participants with established vascular disease or risk factors thereof treated with aspirin ± clopidogrel. Pretreatment PRT and WBA were significantly correlated (collagen r = −.63; arachidonate r = −.65; P < .0001). Following aspirin, the mean PRT increased from 82 to 142 seconds ( P < .0001), and in participants treated with clopidogrel–aspirin, the mean PRT (286 seconds, n = 65) was significantly longer than with aspirin alone (166 seconds, n = 105; P < .001). Only 13% of PRTs of participants treated with clopidogrel and aspirin were within the normal range. We conclude that the PlaCor PRT is a simple, rapid, point-of-care instrument that compares favorably with published descriptions of other platelet function instruments.

AbstractAlthough current antiplatelet therapies provide potent antithrombotic effects, their efficacy is limited by a heightened risk of bleeding and failure to affect vascular remodeling after injury. New lines of research suggest that thrombosis and hemorrhage may be uncoupled at the interface of pathways controlling thrombosis and inflammation. Here, as one remarkable example, studies using a novel and highly selective pharmacologic inhibitor of the spleen tyrosine kinase Syk [PRT060318; 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide] coupled with genetic experiments, demonstrate that Syk inhibition ameliorates both the acute and chronic responses to vascular injury without affecting hemostasis. Specifically, lack of Syk (murine radiation chimeras) attenuated shear-induced thrombus formation ex vivo, and PRT060318 strongly inhibited arterial thrombosis in vivo in multiple animal species while having minimal impact on bleeding. Furthermore, leukocyte-platelet–dependent responses to vascular injury, including inflammatory cell recruitment and neointima formation, were markedly inhibited by PRT060318. Thus, Syk controls acute and long-term responses to arterial vascular injury. The therapeutic potential of Syk may be exemplary of a new class of antiatherothrombotic agents that target the interface between thrombosis and inflammation.

Abstract —Arterial thrombophilia independent of vascular pathology has not been previously defined either experimentally or epidemiologically. To address the existence of an individual propensity to arterial thrombosis, we exploited a previously developed procedure entailing traumatic (crush) injury of paired porcine carotid arteries for generating platelet-rich thrombi. Porcine carotid arteries were injured bilaterally by serial hemostat crushes. Thrombus generation was monitored by local accumulation of autologous 111 In-labeled platelets and Doppler blood flow. Within this cohort of animals of similar age and size, the lowest to the highest responders in thrombus mass spanned a 7-fold range, showing no correlation with shear, platelet or leukocyte count, or plasma concentrations of fibrinogen or von Willebrand factor. However, there was strong intra-individual correlation ( r 2 =0.80; P <0.001) of thrombus deposition between carotid artery pairs. The wide variation in thrombotic response to a standardized stimulus, not accounted for by shear stress or typical hematological variables, appears to be an intrinsic propensity of the individual. The experimental system for thrombus generation is sufficiently quantitative for assessment of variables determining this propensity.