Influence of abciximab on the adhesion of platelets on a shielded plasma gradient prepared on polyethylene

INTRODUCTION: Thrombotic effects of biomaterial implants are mediated merely through activation of the platelet glycoprotein IIb-IIIa (GpIIb-IIIa) receptor. Consequently, platelet GpIIb-IIIa receptor inhibitors are successfully used during stent implantation procedures to prevent thrombosis. However, currently a new generation of stents contains surface coating, which changes the surface to more hydrophobic or hydrophilic. This change markedly affects the interaction of platelets and may influence the efficiency of GpIIb-IIIa inhibitors. MATERIALS AND METHODS: To study the influence of the wettability of biomaterials on the effectiveness of abciximab, 5-cm polyethylene gradients with contact angles of 100 degrees to 40 degrees were made by means of glow discharge. Fresh whole blood with or without abciximab was recirculated over this gradient. RESULTS: Inhibition of platelet adhesion by abciximab was maximal, but not complete, on the hydrophobic and moderate hydrophobic part of the gradient, with contact angles of 55 degrees to 90 degrees. Percentage inhibition by abciximab was maximal around 60 degrees. CONCLUSIONS: Intermediate hydrophobicity of currently applied stent materials, such as stainless steel, seems optimal in combination with abciximab. However, on hydrophobic and particularly on hydrophilic materials, abciximab is less effective.     

The effect of fibrinogen sialic acid residues on ex vivo platelet deposition on biomaterials

The effect of fibrinogen sialic acid residues on platelet deposition onto polymer surfaces was examined using a canine ex vivo shunt model. To test the hypothesis that desialylated fibrinogen may enhance platelet deposition when precoated on biomaterials, canine fibrinogen was desialylated and precoated on polyvinyl chloride (PVC) shunts. When protein-coated PVC shunts were exposed to flowing whole blood, both the native and the desialylated fibrinogen elicited the same profile of platelet deposition. This study indicates that platelet deposition and thrombus formation on biomaterial surfaces is not mediated by a mechanism which involves the sialic acid residues of fibrinogen.     

Antithrombogenic coating of stents using a biodegradable drug delivery technology.

To reduce the thrombogenic properties of coronary artery stents, a biodegradable polylactic acid (PLA) stent coating with an incorporated thrombin inhibitor and a platelet aggregation inhibitor has been developed. In an ex vivo human stasis model, its effect on platelets, plasmatic coagulation and its release characteristics were studied using whole blood. Bare steel and bare gold-surface stents were compared to steel and gold-surface stents coated with PLA (30 kDa) containing 5% polyethyleneglycol (PEG)-hirudin and 1% iloprost, with an empty tube as control. Markers of activated coagulation (prothrombin fragment F1-2 and thrombin-antithrombin III complex, TAT), were assayed and the release of drugs from the coating was assessed by aPTT and collagen-induced platelet aggregation. Bare steel and gold stents were completely covered by a blood clot, and high levels of coagulation markers (F1-2 fragment and TAT) were detected. No differences in the thrombogenic properties were found between bare gold or steel stents. Coated stents were free of blood clots and only minor elevations of markers were detected. Release data from in-vitro studies over 90 days showed a gradual release of the drugs with an initial exponential release characteristic for PEG-hirudin, slow release of iloprost and a 10% degradation of the PLA carrier. This drug releasing biodegradable coating effectively reduced thrombus formation independent of the metallic surface.     

Activation of phosphoipase D by platelet-derived growth factor (PDGF) in rat C6 glioma cells: Possible role in mitogenic signal transduction

Abstract

The effects of platelet-derived growth factor (PDGF) on phosphoiipase D (PLD) activity and deoxyribonucleic acid (DNA) synthesis in rat C6 glioma cells have been investigated. Pretreatment of serum-starved C6 cells with PDGF results in enhanced choline production and the phosphatidylethanol (PEt) formation in the presence of ethanol’ indicating the activation of PLD acting on phosphatidylcholine (PG). The dose-response curve for choline generation and DNA synthesis were comparable. In addition, the effects of PDGF on both PEt formation and [ H]thymidine incorporation into acid-precipitable material was blocked by the potent protein kinase G (PKG) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) but not by N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA1004), a relatively weak inhibitor of PKC, suggesting that PDGF plays an important role as a positive regulator of glioma cell growth via a PLD-mediated mitogenic signal transduction cascades, which depends largely on the activation of PKG.     

生物材料作为口服胰岛素载体的稳定性及缓释作用

摘要 目的：探讨生物材料作为载体,在保持口服胰岛素制备与消化过程中的生物活性及建立满足人体生理需求的胰岛素的释放速率的模式中的应用。 方法：用计算机检索中国期刊全文数据库(CNKI：1989/2009)和Medline database(1989/2009),按纳入和排除标准,对文献进行筛选,资料收集和质量评价,共纳入31篇文章。从常用口服胰岛素载体生物材料、保持口服胰岛素在制备与消化过程中的生物活性及建立满足人体生理需求的胰岛素的释放速率模式3方面进行总结。 结果：以生物材料作为载体包埋胰岛素,可避免胰岛素被胃肠蛋白酶降解,提高其在胃肠道内的稳定性,并有利于肠道黏膜对胰岛素的摄取和转运,在人体内递药过程中又能实现靶向控制药物释放,提高药物的生物利用度,满足人体生理需求的胰岛素的释放速率模式,实现了胰岛素的持续释放。 结论：在胰岛素载体材料的研究过程中,生物材料以其优势发展迅速。随着研究的进一步深入,作为口服胰岛素载体的生物材料将向临床实际应用方向发展。 关键词：口服胰岛素;药物载体;生物材料;壳聚糖;脂质体;聚合物     

Fibrinogen adsorption,platelet adhesion and thrombin generation at heparinized surfaces exposed to flowing blood

Thrombus formation at an artificial surface in contact with blood is a complex process that encompasses accretion of platelets from flowing blood and fibrin deposition. Platelet adhesion and fibrin formation are intimately intertwined reactions that are triggered by different sets of surface adsorbed plasma proteins. To dissect the contribution of protein adsorption and platelet adhesion to thrombin formation, a coherent study was performed with non-coated (NC) and heparin-coated (HC) surfaces. Thrombin production in whole blood, platelet adhesion and protein adsorption were studied using an amidolytic thrombin assay, a dynamic platelet adhesion assay and ellipsometry, respectively. Thrombin generation in flowing whole blood exposed to HC surfaces was greatly diminished when compared with NC surfaces. However, separate platelet adhesion and protein adsorption studies with anticoagulated whole blood revealed that platelets do not adhere because fibrinogen is not available in the protein layer that was deposited during the perfusion. These findings indicate that the in vitro thrombogenicity of a material cannot be predicted from platelet adhesion and protein adsorption data when these measurements are performed with anti-coagulated blood or platelet rich plasma. Preincubation of NC and HC surfaces with fibrinogen or 2000-fold diluted plasma resulted in similar amounts of surface-bound fibrinogen and mediated massive platelet adhesion from flowing whole blood. These results indicate that a) platelet adhesion correlates with the availability of surface-bound fibrinogen and b) NC and HC surfaces are indistinguishable with respect to protein (fibrinogen) adsorption and platelet adhesion. It is apparent that the heparinized surface used in our studies exerts its anti-thrombogenic properties by neutralizing locally formed thrombin and not by reducing fibrinogen-dependent platelet adhesion.     

An in vitro evaluation of the effect of laser irradiation on the thrombogenicity of thrombus.

The effect of laser irradiation on the thrombogenicity of thrombus was evaluated by treating thrombi, formed in-vitro from canine blood, with two different doses of cw Nd:YAG laser energy at 1064 nm. The thrombi were then incubated with whole blood, and the plasma levels of fibrinogen and thrombin-antithrombin III-complexes were measured. A statistically significant decrease (p < 0.05) in the thrombogenicity was indicated by a reduction in both fibrinogen consumption and levels of thrombin-antithrombin III-complexes in the high dose group (600 joules, 100 degrees C peak temperature) in comparison to the low dose group (300 joules, 70 degrees C peak temperature) and the untreated thrombi. These findings suggest that laser irradiation of thrombus at an appropriate dose may substantially reduce its thrombogenicity and ability to modulate hemostasis.     

血浆蛋白对生物材料细菌黏附的影响

生物材料植入感染是生物材料临床应用所面的严重问题，极大地限制着心血管生物材料临床应用和推广，而生物材料细菌黏附是心血管生物材料植入感染的起始动因。因此，防止生物材料细菌黏附是防治生物材料为中心感染的重要环节。研究发现，人体血浆蛋白对生物材料细菌黏附有重要影响，有的血浆蛋白抑制材料上细菌黏附，有的促进材料上细菌黏附。研究血浆蛋白与生物材料细菌黏附关系为防治生物材料为中心感染的发生具有重要意义。     

The influence of synthetic thrombin inhibitors on the thrombin-antithrombin reaction

The reaction of the antithrombin III of blood plasma with thrombin is inhibited in the presence of the reversible inhibitor, 4-amidinophenylpyruvic acid (APPA) and prevented when the active site of thrombin is chemically blocked by reaction with the irreversible inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). These results support the assumption that the thrombin-antithrombin reaction proceeds like an enzyme-substrate reaction.     

Effects of agents, used to treat bleeding disorders, on bleeding time prolonged by a very high dose of a direct thrombin inhibitor in anesthesized rats and rabbits

Melagatran is the active form of the oral, direct thrombin inhibitor, H 376/95, that is under evaluation in clinical trials for the prevention and treatment of thromboembolism. In this study, a single dose, calculated on body weight basis, of antifibrinolytic treatment, factor VIIa, factor VIII with and without von Willebrand factor (vWF), factor IX, activated (APCC) or nonactivated (PCC) prothrombin complex concentrates was given intravenously to rats and rabbits, in an attempt to reverse the prolonged bleeding time during intensive anticoagulation with melagatran (2 micromol/kg/h). The doses used were at or above human therapeutic doses. The cutaneous tail bleeding time in the rat, as well as the ear incision bleeding time and cuticle bleeding time, and the blood loss in the rabbit were used for evaluation of the hemostatic effects of these agents. In vivo Feiba (APCC) and Prothromplex-T (PCC) shortened the prolonged cutaneous bleeding times in rats (P<.05); Feiba and Autoplex (APCC) shortened the cutaneous bleeding times in rabbits (P<.05). In contrast, Prothromplex-T prolonged bleeding times and blood loss in the rabbits (P<.05). Ex vivo Feiba, Autoplex and NovoSeven (rF VIIa) significantly (P<.05) shortened the prolonged whole blood clotting time (WBCT). Prothromplex-T significantly prolonged WBCT, activated clotting time (ACT) and activated partial thromboplastin time (APTT). Feiba, Autoplex, and Prothromplex-T increased thrombin generation measured as increased thrombin-antithrombin complex (TAT) formation. In conclusion, APCCs were found to be the most effective agents for reversing bleeding time induced by a very high plasma concentration of melagatran. APCC and recombinant activated factor FVII (rF VIIa) effectively shortened the prolonged WBCT. Thus, stimulating thrombin generation with the use of APCC may counteract the anticoagulant effect observed with a very high dose of a thrombin inhibitor.     

Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia

Recombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associated with a better therapeutic response as compared to later treatment. In a mouse model it was recently confirmed that recombinant murine (rm-)APC decreases coagulation activation and improves survival in pneumococcal pneumonia; however, APC did not impact on the inflammatory response. The aim of this study was to determine the effect of APC treatment instigated early in infection on activation of coagulation and inflammation after induction of pneumococcal pneumonia. Mice were infected intranasally with viable S. pneumoniae . Mice were treated with rm-APC (125 μg) or vehicle intraperitoneally 12 hours after infection and were sacrificed after 20 hours, after which blood and organs were harvested for determination of bacterial outgrowth, coagulation activation and inflammatory markers. In this early treatment model, rm-APC treatment inhibited pulmonary and systemic activation of coagulation as reflected by lower levels of thrombin-antithrombin complexes and D-dimer. Moreover, rm-APC reduced the levels of a large number of cytokines and chemokines in the lung. When administered early in pneumococcal pneumonia, rm-APC inhibits systemic and pulmonary activation of coagulation and moreover exerts various anti-inflammatory effects in the lung.     

Markers of activated hemostasis and fibrinolysis in patients with pulmonary malignancies: comparison of plasma levels in central venous and pulmonary venous blood

Malignancy frequently is accompanied by activated coagulation and fibrinolysis indicating a hypercoagulable state. The purpose of our study was to estimate the contribution of local tumor-induced mechanisms to the activation of hemostasis and fibrinolysis. In a prospective study, we compared the plasma levels of thrombin-antithrombin complexes, prothrombin fragment 1+2, and D-dimers in blood samples that simultaneously were drawn from the superior vena cava and the pulmonary vein of a tumor-bearing pulmonary lobe. Samples from the superior vena cava were drawn before operation and served as controls. After thoracotomy, a second group of samples was simultaneously taken from the pulmonary veins of the tumor-bearing lobe and the superior vena cava. Forty-five patients with pulmonary malignancies were included (25 adenocarcinomas and 20 squamous cell carcinomas). There were no significant differences of thrombin-antithrombin complexes, prothrombin fragment 1+2, and D-dimers levels in patients suffering from adenocarcinoma and from squamous cell carcinoma. Intraoperatively, prothrombin fragment 1+2 and D-dimers levels were markedly increased when compared with the preoperative values (p<0.0001). There was no increase of thrombin-antithrombin complexes levels due to the operative traumatization. Prothrombin fragment 1+2, thrombin-antithrombin complexes, and D-dimers plasma levels were significantly higher in the pulmonary venous blood than in the blood simultaneously drawn from the superior vena cava (p<0.0001). Our findings indicate that malignant lung tumors directly contribute to the activation of hemostasis and fibrinolysis in these clinical settings.     

Recombinant nematode anticoagulant protein c2, a novel inhibitor of tissue factor-factor VIIa activity,abrogates endotoxin-induced coagulation in chimpanzees

Systemic activation of coagulation leading to disseminated intra-vascular coagulation (DIC) is an important feature in patients with severe sepsis. Tissue factor has been shown to play a primary role in this pathological response, as revealed by the use of specific inhibitors and antagonists of the tissue factor/factor VIIa pathway. This class of agents has been demonstrated to attenuate the coagulation response in human volunteers with induced low-grade endotoxemia and to reduce mortality in primate models of Gram-negative sepsis. The efficacy of these agents in attenuating the activation of coagulation and formation of microvascular thrombosis in sepsis may depend on the mechanism of inhibition. Here we demonstrate the efficacy of recombinant nematode anticoagulant protein c2 (rNAPc2) that specifically inhibits the tissue factor/factor VIIa complex by a novel mechanism, in a model of endotoxin-induced coagulation activation in chimpanzees. Administration of a low dose of Gram-negative endotoxin induced marked increases of thrombin generation as measured by plasma levels of prothrombin activation fragment F(1+2) and thrombin-antithrombin complexes, which were completely blocked by rNAPc2. In chimpanzees receiving rNAPc2 alone, there was a significant reduction in the activation of factor X but not factor IX, compared to animals receiving placebo. In contrast to the effect of rNAPc2 on thrombin generation, there was no effect of this inhibitor on the well known enhanced systemic fibrinolytic response induced by endotoxin. In conclusion, the recombinant peptide rNAPc2 is an effective inhibitor of tissue factor-driven thrombin generation during low grade endotoxemia. These results suggest that rNAPc2 may be a promising therapeutic option to inhibit coagulation activation in patients with sepsis.     

Calibrated automated thrombin generation in normal uncomplicated pregnancy

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.     

Thrombin formation in vitro in response to shear-induced activation of platelets

INTRODUCTION: Thromboembolic events caused by implanted vascular devices present serious medical challenges. In particular bileaflet mechanical heart valves (MHVs) are prone to thrombus formation in the hinge region due to a combination of high shear stress and stagnation regions. Most studies of shear-induced platelet activation and aggregation have been performed using viscometers, parallel plate flow, and other non-physiologic in vitro configurations. The present study investigated these events in a physiogically relevant environment in which thrombin formation in response to shear stress activation of platelets plays a more predominant role. MATERIALS AND METHODS: Anticoagulated (citrated) human blood was placed in a steady flow loop containing a 400 microm round orifice or various MHVs in the leakage position. Simultaneous blood recalcification enhanced the thrombus forming potential of the blood. Aggrastat and AN51 were used to block binding to the platelet GPIIb/IIIa and GPIb receptors, respectively, and aspirin was used to block thromboxane production. Thrombin generation was measured indirectly by the thrombin-antithrombin III assay. RESULTS AND CONCLUSIONS: Aggrastat, AN51, and aspirin all suppressed thrombin formation. Furthermore, histological results suggested important roles for vWF and fibrinogen in a two-step model of thrombus formation. Thus, thrombin is reproducibly formed in this in vitro system, a process that can be suppressed by blocking platelet activation. This system has the potential to investigate mechanisms and interventions for medical devices that contact with blood under varying shear stress conditions.     

Thrombin generation in patients after acute deep-vein thrombosis

Thrombin generation measurement may be of value for assessing the risk of venous thromboembolism, but its long term profile has not been assessed in patients. We evaluated thrombin generation by Calibrated Automated Thrombogram (CAT) in plasma during follow up of 104 consecutive patients after an acute episode of deep venous thrombosis. Blood was drawn three times over the course of 24 months. Thrombin generation was measured in absence and presence of thrombomodulin and compared to a reference range derived from thrombin generation curves in 137 healthy volunteers. Thrombin generation of patients showed significantly higher endogenous thrombin potential (ETP) and peak height compared to the reference population. Differences were more pronounced in assays triggered with 1 pM TF. Inhibition by thrombomodulin was attenuated in patients off anticoagulants as compared to the reference population (21% vs. 42.2%, p < 0.0001); inhibition in patients on anticoagulant treatment was less pronounced (9.7%, p < 0.0001). Protein C activity, protein S antigen as well as free protein S showed highly negative correlation with ETP in all patients. A significant negative relation was found between FVIII levels and thrombomodulin induced reduction of ETP and peak height. In conclusion, thrombin generation by CAT reflects changes in coagulation status in patients following a thromboembolic event and is most sensitive at CAT analysis triggered with 1 pM TF. A role for factor VIII as an important attributable cause of hypercoagulability is reflected by the reduced inhibitory effect of thrombomodulin at high factor VIII levels.     

重症肌无力中枢神经系统损害与乙酰胆碱酯酶关系的研究

目的　研究重症肌无力 (MG)中枢神经系统 (CNS)损害大鼠模型乙酰胆碱酯酶 (AChE)的变化。方法　将MG患者血中提取的IgG注入大鼠脑室系统 ,致大鼠CNS损害 ,然后分别观察实验组和对照组大鼠脑、血中AChE的变化。结果　脑室注射后脑组织中AChE水平升高 ,与对照组比较差异显著 (P <0 .0 1) ,而血中AChE水平无明显变化。结论　MG患者IgG致大鼠CNS损害时脑组织AChE水平显著升高 ,表明MG患者CNS损害与CNS胆碱能神经系统功能障碍有关。     

Improved blood compatibility of a stent graft by combining heparin coating and abciximab

The aim of this study was to assess the combined effect of heparin coating of a stent graft and administration of abciximab, on platelet and coagulation activity in vitro. METHODS: Stent grafts with an expanded polytetrafluoroethylene (ePTFE) membrane interfoliated between two stents were deployed in tubings to form Chandler loops. Fresh human blood with a low concentration of heparin and various doses of abciximab was rotated for 1 h, then collected and used for measurements of platelets, thrombin-antithrombin complex (TAT), CD11b, complement and contact activation of coagulation. In the first set of experiments, all stent grafts were heparin coated. There were three study groups: Group 1a (no abciximab, n=5); Group1b (abciximab in a concentration of 3.3 microg/ml, n=5); Group 1c (abciximab in a concentration of 8.3 microg/ml, n=5). In the second set of experiments, the concentration of abciximab was 3.3 microg/ml. There were three study groups: Group 2a (untreated stent grafts, n=4); Group 2b (heparin-coated stent grafts, n=4); Group 2c (heparin-coated PVC tubings with no stent grafts, n=4). RESULTS: In the first set of experiments, there was a significant reduction in platelet count in Group 1c compared to Group 1a and Group 1b. There was a significant reduction in TAT in Group 1b and Group 1c as compared to Group 1a. In the second set of experiments, TAT was reduced in Group 2b and Group 2c compared to Group 2a. Contact activation was lowered in Group 1b and Group 1c as compared to Group 1a for both FXIa-AT (0.088 and 0.088 vs. 0.115) and FXIIa-AT (0.12 and 0.12 vs. 0.19). CONCLUSION: Heparin coating of a stent graft was shown to improve blood compatibility and this was further enhanced by addition of abciximab.     

输血系统生物材料的研究进展及其生物相容性☆

摘要：随着输血材料研究的深入，输血不良反应的发生逐渐减少，人们对输血的认识也在不断提高。医用生物材料与血液间接或直接接触，将对血液中血小板、红细胞、白细胞及血浆蛋白等成分发生作用，相互作用的结果导致血栓形成、溶血、补体系统激活及血液中有形成分改变。医疗器械的血液相容性评价是医用生物材料生物学评价的重要组成部分。文章通过介绍输血技术的研究进展，自身输血为何能得到广泛应用，进而阐述了3种自身输血方法的涵义，各种输血方法在临床的应用，以及常见临床输血不良反应的原因。     

Treatment of Hyperhomocysteinemia with Folic Acid and Vitamins B12 and B6 Attenuates Thrombin Generation

The effect of homocysteine-lowering treatment on thrombin generation was investigated in 17 subjects with hyperhomocysteinemia (aged 22–60 years), 11 of whom had symptomatic atherosclerotic vascular disease. All subjects had fasting total homocysteine levels above 16 μmol/L. The formation of thrombin was assessed by measuring thrombin-antithrombin III complexes and prothrombin fragment 1+2 in peripheral venous blood and in the bleeding time blood collected at 30-second intervals from skin incisions on a forearm. All the tests were performed before and after an 8-week treatment with folic acid p.o. 5 mg/day, vitamin B₆ p.o. 300 mg/day, and vitamin B₁₂ i.m. 1000 μg given on a weekly basis. Following the 8-week therapy, the median plasma homocysteine concentration became significantly reduced from 20 to 10 μmol/L, while plasma levels of fibrinogen, prothrombin, and antithrombin III as well as activity of protein C, S, and factor VII showed no changes. Vitamin treatment was associated with a significant fall in thrombin-antithrombin III complexes and prothrombin fragment 1+2 concentrations in peripheral venous blood. Bleeding time became prolonged by about 60 seconds. At sites of hemostatic plug formation, plasma concentrations of both thrombin markers significantly decreased. Compared with pretreatment values, significantly less thrombin was produced during the first 3 minutes of bleeding after homocysteine-lowering therapy. In subjects with hyperhomocysteinemia a reduction of plasma fasting homocysteine concentration by folic acid and vitamins B₁₂ and B₆ administration is associated with attenuation of thrombin generation both in peripheral blood and at sites of hemostatic plug formation.