冻伤对大鼠血凝系统某些因素的影响

本研究观察了大鼠双后足局部冻伤后其血凝系统某些因素的改变。结果表明,冻伤后大鼠的出血时间及凝血时间明显缩短,血小板数增加,血小板聚集率增高,血浆血栓素Ａ２、纤维蛋白原及钙离子浓度均明显增加,且上述指标的改变与冻伤程度有密切关系,冻伤愈重,改变愈明显,恢复亦愈慢。结果提示,冻伤可使血凝系统发生改变,血液处于高凝状态,这对浆伤后发生的血液循环障碍最终导致冻伤组织坏死的病理过程起重要作用。     

蝮蛇抗栓酶对冻伤家兔血凝系统某些指标的影响

本研究观察了家兔双后足重度冻伤后用蝮蛇抗栓酶（０．２５Ｕ／ｋｇ体重加入２０ｍｌ／ｋｇ体重生理盐水中,耳缘静脉点滴）进行治疗后其血凝系统某些指标的改变。结果表明,重度冻伤后家兔的出血时间及凝血时间明显缩短,血小板功能呈不同程度的改变,血浆纤维蛋白原含量增加,血液处于高凝状态;用蝮蛇抗栓酶治疗后,上述各项指标均有不同程度的改善,结果提示,蝮蛇抗栓酶可明显缓解冻伤所致的血凝增强的病变过程,这对重度冻伤的治疗将起到重要作用     

Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis C.H. Wright in vitro and in vivo

Thrombus formation in blood vessel plays an important role in the pathogenesis and progression of atherosclerosis and cardiovascular diseases. Extract of Dioscorea zingiberensis C.H. Wright (D. zingiberensis) is demonstrated to posses activities for curing cardiovascular diseases such as coronary heart disease and angina pectoris. However, there were few studies on anti-thrombosis activity of it. We investigated the anti-thrombosis effect of diosgenin from D. zingiberensis (Dio) in vitro and in vivo on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. We evaluated the protective effect of Dio by measuring the platelet aggregation, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and the venous thrombosis in rats and the bleeding time, clotting time and protection rate in mice. Results showed that Dio inhibited platelet aggregation, thrombosis and prolonged APTT, PT and TT in rats in a dose-dependent manner. They also prolonged the bleeding time, clotting time and increased protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that Dio which contained 95% diosgenin had anti-thrombosis activity. Dio executives the anti-thrombosis activity through improving the anticoagulation function, inhibiting platelet aggregation and thrombosis.     

Changes in thromboxane A2 generation and plasma lipid pattern in pseudomenopause induced by gonadotropin releasing hormone (GnRH) analogue buserelin

An increased risk of cardiovascular disease has been found in postmenopausal women in comparison to premenopausal women. The aim of this study was to investigate platelet function, blood clotting and plasma lipid levels in 12 women with a condition of hypoestrogenism, similar to the postmenopausal status induced by treatment with the GnRH analogue buserelin for uterine leiomyoma. Platelet aggregation in whole blood and platelet-rich plasma (PRP), serum thromboxane (TX) B2 production, fibrinopeptide A (FPA) plasma levels and plasma lipid pattern were measured before and after 13 weeks of buserelin treatment. No changes of platelet aggregability were found either in whole blood or PRP. Serum TXB2 generation increased significantly after 13 weeks of therapy (p less than 0.001). No signs of increased thrombin generation were found, as indicated by unchanged FPA plasma levels. Total cholesterol plasma levels were found increased after 13 weeks, LDL cholesterol levels showed a tendency to increase although not significantly. HDL cholesterol and triglyceride concentrations were unaffected. The changes of arachidonic acid metabolism and lipid pattern suggest that buserelin treatment may induce a condition of increased thrombotic risk even if the lack of enhanced thrombin generation and increased platelet aggregability indicates that no blood clotting activation occurs.     

Effect of omega-3 fatty acids on haemostatic functions in urocortin-treated obese rats

Urocortin 1 (UCN1) decreases food intake. We investigated the effects of UCN1 and omega-3 fatty acids (FA) on metabolic and coagulation parameters in high fat diet (HFD)-fed rats. Fifty male Sprague Dawley rats were divided into five groups; control, HFD, HFD with omega-3 FA, HFD with UCN1, and HFD with UCN1 and omega-3 FA. Food intake, body weight (BW), body mass index (BMI), Lee index, glucose, insulin, HOMA-IR, triglycerides, cholesterol, low (LDL) and high (HDL) density lipoproteins, fibrinogen, plasminogen activator inhibitor 1 (PAI-1), fibrin degradation product (FDP), clotting time, bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), and platelet aggregation were measured. Food intake, BW, BMI, Lee index, glucose, insulin, HOMA-IR, triglycerides, cholesterol, LDL, fibrinogen, platelet aggregation, PAI-1, and FDP increased while bleeding and clotting times, PT, and aPTT decreased in HFD rats. UCN1 decreased food intake, BW, BMI, Lee index, bleeding and clotting times, PT, and aPTT and increased fibrinogen, PAI-1, FDP, and platelet aggregation in HFD rats. Omega-3 FA decreased food intake, BW, BMI, Lee index, platelet aggregation, glucose, insulin, HOMA-IR, triglycerides, and increased HDL and bleeding time in HFD rats. We concluded that UCN1 worsens the hypercoagulable state in HFD rats while omega-3 FA improve the insulin resistance and decrease the platelet aggregation in those rats.     

Analogy between fibrinogen and casein. Effect of an undecapeptide isolated from kappa-casein on platelet function

A large number of similarities have previously been noted between the blood and milk clotting phenomena [Jollès, P. (1975) Mol. Cell. Biochem. 7, 73-85; Jollès, P. & Henschen, A. (1982) Trends Biochem. Sci. 7, 325-328]: some analogous features have also been found between fibrinogen and kappa-casein. In this connection, the effect of a natural and a synthetic peptide derived from kappa-casein on platelet function was studied: the undecapeptide Met-Ala-Ile-Pro-Pro-Lys-Lys-Asn-Gln-Asp-Lys (residues 106----116 of cow kappa-casein) inhibited both aggregation of ADP-treated platelets and binding of 125I-fibrinogen to ADP-treated platelets: its behaviour was similar to that of the structurally related C-terminal dodecapeptide of human fibrinogen gamma-chain.     

Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin alpha IIb beta 3.

Blood loss at sites of vascular rupture is controlled by the adhesion and aggregation of platelets and the formation of an insoluble fibrin matrix. Fibrinogen is considered to be critical in these processes by both providing an abundant dimeric ligand for alpha IIb beta 3-mediated platelet aggregation, and serving as the fundamental building block of the fibrin polymer. To establish an in vivo model system to examine in detail the importance of alpha IIb beta 3-fibrinogen interactions in platelet function, hemostasis, response to injury and vasoocclusive disease, and to test the prevailing hypothesis that the C-terminal segment of the fibrinogen gamma chain is essential for alpha IIb beta 3 binding, we have used gene-targeting technology in mice to eliminate the last five residues (QAGDV) from the gamma chain. Mice homozygous for the modified gamma chain gene (gamma delta 5/gamma delta 5) displayed a generally normal hematological profile, including normal platelet count, plasma fibrinogen level, clotting time and fibrin crosslinking. However, both gamma delta 5-fibrinogen binding to alpha IIb beta 3 and platelet aggregation were highly defective. Remarkably, another alpha IIb beta 3-dependent process, clot retraction, was unaffected by the gamma delta 5 mutation. Despite the preservation of clotting function, gamma delta 5/gamma delta 5 mice were unable to control blood loss following a surgical challenge and occasionally developed fatal neonatal bleeding events.     

Coumarin Therapy and Platelet Aggregation

Platelet aggregation has been related to blood coagulation studies in patients on nicoumalone, a coumarin anticoagulant. Aggregation studies were performed by means of Chandler''s tube and the adenosine diphosphate (A.D.P.)-induced optical density method. Platelet aggregation in Chandler''s tube has been shown to be quite different from A.D.P. aggregation and to be dependent on the “intrinsic” (blood) clotting system. When the intrinsic system was depressed by coumarin anticoagulant, aggregation was delayed in Chandler''s tube, but patients with a predominantly “extrinsic” (tissue) system defect gave normal results even when their prothrombin time was excessively prolonged. In contrast there was an increased response to A.D.P. in the anticoagulated patients.The study emphasizes the different mechanisms of platelet aggregation, which we have referred to as coagulation-induced and A.D.P.-induced aggregation. It also shows the limitations of routine control of oral anticoagulants by prothrombin time alone, as the coagulation-induced platelet aggregation appears to be quantitatively related to the overall level of clotting factors in the intrinsic system and independent of the extrinsic system.     

Thrombin-like serine protease,antiquorin from Euphorbia antiquorum latex induces platelet aggregation via PAR1-Akt/p38 signaling axis

Plant latex proteases (PLPs) are pharmacologically essential and are integral components of traditional medicine in the management of bleeding wounds. PLPs are known to promote blood coagulation and stop bleeding by interfering at various stages of hemostasis. There are a handful of scientific reports on thrombin-like enzymes characterized from plant latices. However, the role of plant latex thrombin-like enzymes in platelet aggregation is not well known. In the present study, we attempted to purify and characterize thrombin-like protease responsible for platelet aggregation. Among tested plant latices, Euphorbia genus latex protease fractions (LPFs) induced platelet aggregation. In Euphorbia genus, E. antiquorum LPF (EaLPF) strongly induced platelet aggregation and attenuated bleeding in mice. The purified thrombin-like serine protease, antiquorin (Aqn) is a glycoprotein with platelet aggregating activities that interfere in intrinsic and common pathways of blood coagulation cascade and alleviates bleeding and enhanced excision wound healing in mice. In continuation, the pharmacological inhibitor of PAR1 inhibited Aqn-induced phosphorylation of cPLA₂, Akt, and P38 in human platelets. Moreover, Aqn-induced platelet aggregation was inhibited by pharmacological inhibitors of PAR1, PI3K, and P38. These data indicate that PAR1-Akt/P38 signaling pathways are involved in Aqn-induced platelet aggregation. The findings of the present study may open up a new avenue for exploiting Aqn in the treatment of bleeding wounds.     

Effects of repeated oral doses of fenflumizole on platelet aggregation and thromboxane formation in man ex vivo

Anti-platelet effects of fenflumizole, a new cyclo-oxygenase inhibitor, were studied in man ex vivo. Fenflumizole was given to male volunteers at the oral doses of 25, 50 or 100 mg per day, each dose for a period of seven days. The formation of thromboxane B2 (TXB2) during whole blood clotting, platelet aggregation induced by arachidonic acid and ADP, the formation of TXB2 during aggregation as well as serum concentration of fenflumizole were measured repeatedly during drug administration and for a fortnight after drug discontinuation. TXB2 formation during whole blood clotting was decreased dose-dependently by fenflumizole. The degree of inhibition of TXB2 formation was proportional to fenflumizole concentration in serum within each individual. The lag phase of platelet aggregation induced by arachidonic acid was prolonged and the formation of TXB2 during aggregation decreased by fenflumizole. No total inhibition of either TXB2 synthesis or platelet aggregation was caused by the fenflumizole doses used. The results show that the degree of inhibition of platelet thromboxane forming capacity by repeated doses of fenflumizole is closely related to the concentration of the drug in blood. Platelet aggregation however is less sensitive to changes in fenflumizole levels and cannot be assessed solely on the basis of cyclo-oxygenase activity.     

Effects of dietary palm oil on serum lipid peroxidation, antithrombin III, plasma cyclic AMP, and platelet aggregation.

Intravascular thrombus formation in association with lipid depositions in the arterial wall is thought to be involved in the process of atheroma formation. We have previously shown the beneficial effect of palm oil on the serum lipid profile resulting in a lowering of serum triacylglycerol and an elevation of the HDL/LDL ratio. The present study investigates the effect of dietary palm oil on the biochemical parameters associated with clotting and platelet aggregation in young rats (70 g body wt) fed a palm oil diet over a period of 10 weeks. Palm oil-fed rats showed significantly lower levels of fibrinogen and serum lipid peroxide and elevated AtIII levels resulting in a prolongation of clotting time. Reduced platelet aggregation and ATP release associated with a prolongation of bleeding time were also found. These findings, together with our earlier findings on the effect of palm oil on the serum lipid profile, suggest that dietary palm oil may be antithrombotic as well as beneficial in preventing the deposition of lipids on the vessel wall and may, therefore, be protective against the development of atherosclerosis.     

Glycine reduces platelet aggregation

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl^? influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1–10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.     

The interaction of β2-glycoprotein I with lysophosphatidic acid in platelet aggregation and blood clotting

β₂-Glycoprotein I (β₂-GPI) is a plasma protein that binds to oxidized low-density lipoprotein (LDL) and negatively charged substances, and inhibits platelet activation and blood coagulation. In this study, we investigated the interaction of β₂-GPI with a negatively charged lysophosphatidic acid (LPA) in platelet aggregation and blood clotting. Two negatively charged lysophospholipids, LPA and lysophosphatidylserine, specifically inhibited the binding of β₂-GPI to oxidized LDL in a concentration-dependent manner. Intrinsic tryptophan fluorescence studies demonstrated that emission intensity of β₂-GPI decreases in an LPA-concentration-dependent manner without a shift in wavelength maxima. LPA specifically induced the aggregation of β₂-GPI in phosphate-buffered saline, and in incubated plasma and serum, both of which are known to accumulate LPA by the action of lecithin-cholesterol acyltransferase and lysophospholipase D/autotaxin. β₂-GPI aggregated by LPA did not inhibit activated von Willebrand factor-induced aggregation, and did not prolong the activated partial thromboplastin time in blood plasma, in contrast to non-aggregated β₂-GPI. These results suggest that β₂-GPI aggregated by the binding to LPA fails to inhibit platelet aggregation and blood clotting in contrast to non-aggregated β₂-GPI.     

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.     

Influence of propranolol on platelet aggregation and thromboxane B2 production from platelet-rich plasma and whole blood

The beta-adrenoceptor antagonist propranolol is used in the therapy of hypertension and ischemic heart disease. The aim of our study was to evaluate the effects of this drug on platelet aggregation and on synthesis of thromboxane B2 (the stable metabolite of Thromboxane A2) from platelet rich plasma (PRP), whole blood samples and during spontaneous clotting. The results indicate that propranolol at concentrations near the therapeutic range, significantly inhibit collagen and thrombin-induced platelet aggregation and TxB2 synthesis from PRP. Furthermore the drug demonstrates inhibitory activity on B-TG release and TxB2 production from whole blood samples and on spontaneous clotting. The results suggest that some benefits of propranolol in the treatment of patients with coronary artery disease or cardiovascular conditions associated with platelet hyperaggregability may also be related to interference with platelet activation "in vivo" and with TxA2 generation.     

C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity

We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.     

Platelet aggregation and ATP secretion in whole blood of normal cats and cats homozygous and heterozygous for Chediak-Higashi syndrome

A rapid and simple technique using the Whole Blood Lumi-Aggregometer was used to study storage pool disease in Chediak-Higashi homozygote and heterozygote cats. Feline Chedlak-Higashi platelets aggregated after the addition of both ADP and collagen. During platelet aggregation, ATP secretion was assayed; the whole blood aggregometer is effective in detecting decreased levels of secretable ATP in homozygote cats. No storage pool deficiency was found in heterozygote cats. However, upon analysis of impedance tracings, a decreased platelet aggregation response was seen in both homozygote and heterozygote cats. These results suggest that prolonged bleeding times in Chediak-Higashi cats may involve a mechanism in addition to a dense granule deficiency.     

An ethanol extract of Ramulus mori improves blood circulation by inhibiting platelet aggregation

Inappropriate platelet aggregation can cause blood coagulation and thrombosis. In this study, the effect of an ethanol extract of Ramulus mori (ERM) on blood circulation was investigated. The antithrombotic activity of ERM on rat carotid arterial thrombosis was evaluated in vivo, and the effect of ERM on platelet aggregation and blood coagulation time was evaluated ex vivo. To evaluate the safety of ERM, its cytotoxicity to platelets and its effect on tail bleeding time were assessed; ERM was not toxic to rat platelets and did not prolong bleeding time. Moreover, administering ERM to rats had a significant preventive effect on carotid arterial thrombosis in vivo, and significantly inhibited adenosine diphosphate- and collagen-induced platelet aggregation ex vivo, whereas it did not prolong coagulation periods, such as prothrombin time and activated partial thromboplastin time. The results suggest that ERM is effective in improving blood circulation via antiplatelet activity rather than anticoagulation activity.     

OKY-1581: A selective inhibitor of thromboxane synthesis and

OKY-1581 is an effective inhibitor of thromboxane synthesis and . The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either or caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Newly Identified HNP‐F from Human Neutrophil Peptide‐1 Promotes Hemostasis

Active hemostatic agents can play a crucial role in saving patients’ lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide‐1 (HNP‐1) has been previously reported with the hemostatic mechanism, which part of HNP‐1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP‐F) promoting hemostasis, originating from HNP‐1, has been newly identified by the blood coagulation ability test. HNP‐F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP‐F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP‐F's biosafety. It is hypothesized that the surface charge and structure of HNP‐F could be favorable to interact with fibrinogen or thrombospondin‐1. Collectively, because HNP‐F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.