应用阿加曲班在体外抑制针对胰岛的即刻经血液介导的炎性反应

目的 探讨凝血酶抑制剂--阿加曲班对胰岛移植后即刻经血液介导的炎性反应(IBMIR)的抑制作用.方法 分离、纯化Wistar大鼠胰岛,采用体外循环模型模拟IBMIR,设立空白对照组、对照组与实验组,对照组将血液与胰岛直接混合,实验组在对照组的基础上加入阿加曲班注射液,空白对照组只加入血液,而无胰岛.各组于37℃下循环反应60 min后,将内容物通过70 tan滤网过滤,将对照组与实验组的过滤残余血栓块和组织经膜式薄层细胞检测(TCT),滤过液进行血常规检测,并以胰岛素释放试验判定其中胰岛的功能.结果 实验组滤过液中的血小板数、白细胞数、单核细胞数和淋巴细胞百分数显著高于对照组,差异均有统计学意义(P＜0.05).实验组的胰岛在高糖和低糖的刺激下,胰岛素释放量明显高于对照组,其胰岛素释放指数为3.36±0.18,明显高于对照组的2.25±0.18(P＜0.05).对照组过滤残余的血栓块和组织中胰岛数量较少,结构破坏严重,被膜不完整,胰岛周嗣见大量红细胞形成的微血栓包绕,实验组中胰岛数量较多,结构完整.呈团块状,胰岛周围未见明显微血栓形成.结论 阿加曲班在体外可以有效抑制针对胰岛的IBMIR,减轻对胰岛细胞的损伤.     

The effects of aprotinin and tranexamic acid on thrombin generation and fibrinolytic response after cardiac surgery

Background: Thrombin formation during cardiac surgery could result in disordered hemostasis and thrombosis. The aim of the study was to examine the effects of aprotinin and tranexamic acid on thrombin generation and fibrinolytic activity in patients undergoing cardiac surgery. Methods: Data were collected prospectively from 60 patients undergoing coronary artery bypass grafting using cardiopulmonary bypass (CPB). In a randomized sequence, 20 patients received aprotinin, 20 patients received tranexamic acid, and in 20 patients placebo was used. Results: Significant thrombin activity was found in all the studied patients. Thrombin generation was less in the aprotinin group than in the tranexamic acid and the placebo group (thrombin/anti‐thrombin III complexes 33.7 ± 3.6, 53.6 ± 7.0 and 44.2 ± 5.3 µg/l 2 h after CPB and F1 + 2 fragment 1.50 ± 0.10, 2.37 ± 0.37 and 2.04 ± 0.20 nmol/l 6 h after surgery, respectively). The inhibition of fibrinolysis was significant with both anti‐fibrinolytic drugs (d ‐dimers 0.427 ± 0.032, 0.394 ± 0.039 and 2.808 ± 0.037 mg/l 2 h after CPB, respectively). The generation of d ‐dimers was inhibited until 16 h after CPB in the aprotinin group. The plasminogen activation was significantly less in the aprotinin group (plasmin/anti‐plasmin complexes 0.884 ± 0.095, 2.764 ± 0.254 and 1.574 ± 0.185 mg/l 2 h after CPB, respectively). Conclusion: Thrombin formation is inevitable in coronary artery bypass surgery when CPB is used. The suppression of fibrinolytic activity, either with aprotinin or with tranexamic acid interferes with the hemostatic balance as evaluated by biochemical markers. Further investigations are needed to define the role of hemostatic activation in ischemic complications associated with cardiac surgery.     

人凝血酶在手术中止血作用的随机对照和多中心临床研究

目的评价人凝血酶在外科手术中的止血作用和安全性。方法采用前瞻、随机、对照和多中心的方法 ,针对腹部手术切口 96例和肝脏手术切面 4 0例两种创面分别进行研究 ,每种创面均等分为 2组 ,研究组使用凝血酶 (切口 10 0 0IU ,肝创面 2 0 0 0IU) ,对照组给予等量生理盐水。对手术创面出血的止血时间、出血量、单位面积出血量以及肝手术患者术后 2 4h腹腔引流量等方面和安全性进行观察。结果研究组腹部切口创面止血时间为 ( 10 4± 70 )s ,单位面积出血量为 ( 0 4± 0 3)g/cm2 ,在肝创面分别为 ( 10 6± 78)s、( 1 0± 0 7)g/cm2 ,与对照组结果相比差异有显著性意义 (P <0 0 1) ,术后腹腔引流量研究组为 ( 10 3± 2 8)ml,明显少于对照组的 ( 2 0 4± 15 9)ml(P <0 0 5 )。结论人凝血酶对腹部切口和肝创面毛细血管出血有较好的止血作用和安全性。     

琥珀酰明胶影响凝血酶生成的机制

目的: 探讨琥珀酰明胶（gelatin, GEL）影响机体凝血酶生成的相关机制。方法: 选取12例健康志愿者,采集外周静脉血,分别加入GEL（GEL组）和乳酸钠林格液（lactated Ringer's solution, LR）（LR组）。以基础值、10∶2、10∶4和10∶6的稀释度进行血液稀释。观察GEL和LR对凝血因子活性和血小板功能的影响。结果: 两组各凝血因子活性随着稀释度增加而明显下降。但在相同稀释条件下,两组间各凝血因子活性差异无统计学意义。GEL降低血小板P选择素的表达,但不影响血小板糖蛋白Ⅱb/Ⅲa受体和血小板磷脂酰丝氨酸的表达以及血小板体积。GEL对血小板计数的影响也与LR一致。结论: GEL通过降低血小板P选择素的表达,影响凝血酶在爆发阶段的生成,导致凝血酶生成的速度和峰值均降低。     

The effects of aprotinin on hemostatic function during cardiac surgery.

The mechanism of action by which large doses of aprotinin decrease blood loss during cardiac surgery is not completely understood. In a prospective, controlled study, 30 patients undergoing cardiac surgery were given high-dose aprotinin in accordance with a commonly used regimen. Twenty untreated but otherwise comparable patients served as the control group. The effects of aprotinin therapy during cardiopulmonary bypass on coagulation parameters, the kallikrein-kinin system, fibrinolysis, platelet stimulation, and the release of elastase from neutrophils were studied. The fibrinolysis parameters were the only measurements that showed clear and significant differences between the two groups. Aprotinin almost completely inhibited the formation of fibrin and fibrinogen degradation products. It is assumed that inhibition of systemic fibrinolysis and suppression of local fibrinolysis contribute to the hemostatic action of aprotinin. The study did not demonstrate a significant protective effect of aprotinin on platelets. In addition, the dose of aprotinin administered did not affect the kallikrein-kinin system of elastase. Therefore, these data suggest that the previously demonstrated hemostatic effects of aprotinin derive primarily from its antifibrinolytic action.     

Systemic effects of tissue plasminogen activator-associated fibrinolysis and its relation to thrombin generation in orthotopic liver transplantation

Orthotopic liver transplantation is frequently associated with hyperfibrinolysis, the origin and clinical relevance of which is largely unknown. In 20 orthotopic liver transplantations, we studied the occurrence and systemic effects of hyperfibrinolysis. Severe fibrinolysis was defined to be present when the euglobulin-clot lysis time and the whole-blood-clot lysis time, as measured by thrombelastography, were shorter than 60 and 90 min, respectively, at some time during the operation. Based on these criteria, 7 patients had minimal fibrinolysis (group I), and 13 patients had severe fibrinolysis (group II). In group II a gradual increase of tissue-type plasminogen activator (t-PA) activity was seen during the anhepatic stage, followed by an "explosive" increase immediately after graft reperfusion (P = 0.0004, compared with group I), and a reduction of plasminogen activator inhibitor (PAI) activity. Plasma degradation products of fibrinogen and fibrin increased parallel to t-PA activity, and levels were significantly higher at 45 min after graft reperfusion in group II compared with group I (P less than 0.04). Thrombin-antithrombin III complexes showed an identical steady increase in both groups, indicating that increased t-PA activity was not related to thrombin formation. A combination of increased endothelial release and reduced hepatic clearance may have caused the increased t-PA activity. The t-PA-associated destruction of fibrinogen and fibrin after graft reperfusion is consistent with the clinical signs of severe oozing often seen in this period. These observations may have important clinical implications for the treatment of bleeding in patients undergoing orthotopic liver transplantation.     

凝血酶对骨髓基质干细胞功能的影响

目的 探讨凝血酶对骨髓基质干细胞功能的影响.方法 小鼠骨髓基质干细胞取自于4周龄C57BL/6J小鼠双侧胫骨和股骨的新鲜骨髓,细胞增殖的检测采用5-溴-2-脱氧尿嘧啶嵌入测定试剂盒,细胞迁移的测定通过细胞计数法,细胞凋亡是根据4,6-二氨基-2-苯基吲哚所染细胞核形态的改变来辨认.结果 凝血酶可以促进骨髓基质干细胞剂量依赖性的增殖、抑制细胞的凋亡,但不影响细胞的迁移.结论 结果表明凝血酶对骨髓基质干细胞增殖和凋亡均有影响,这种影响可能有助于保障组织修复过程中所需的细胞的数量,这种功能可能是凝血酶在组织损伤修复过程中起保护作用.     

A comparison of recombinant thrombin to bovine thrombin as a hemostatic ancillary in patients undergoing peripheral arterial bypass and arteriovenous graft procedures

OBJECTIVES: Recombinant thrombin (rThrombin) is a potential hemostatic alternative to bovine and human plasma-derived thrombin. This report examines the clinical results for the vascular surgery subgroup of patients enrolled in a larger double-blind, randomized, multicenter trial, which evaluated the comparative safety and efficacy of rThrombin and bovine plasma-derived thrombin (bThrombin) when used as adjuncts to surgical hemostasis. METHODS: Data from the 164 vascular patients who underwent either a peripheral arterial bypass (PAB) or arteriovenous graft (AV) procedure are included in this analysis. Time to hemostasis at proximal and distal anastomotic sites at 1.5-, 3-, 6-, and 10-minute intervals was determined by procedure (PAB or AV) and overall (PAB + AV). Baseline and day 29 immunologic sera were analyzed. The incidences of postoperative adverse events were compared between treatment groups. Categorical adverse events were evaluated in relation to thrombin product antibody formation. RESULTS: Patients were randomized to either bThrombin (n = 82) or rThrombin (n = 82). Procedures included PAB (n = 88) and AV (n = 76). The bThrombin and rThrombin groups were well matched for demographics and baseline characteristics. A comparable incidence of anastomotic hemostasis was observed in both treatment groups at 10 minutes (94% bThrombin, 91% rThrombin). The incidence of hemostasis was lower at all time points for PAB procedures compared with AV procedures. In the PAB group, a significantly greater proportion of patients receiving rThrombin (55%) achieved hemostasis at 3 minutes compared with bThrombin (39%; P < .05). Adverse event profiles and laboratory findings were similar between groups. No patients in the rThrombin group developed anti-rThrombin product antibodies at day 29, whereas 27% of patients in the bThrombin group developed antibodies to bThrombin product (P < .0001). CONCLUSIONS: rThrombin or bThrombin used as a hemostatic ancillary for anastomotic bleeding was equally effective at 10 minutes; however, rThrombin compared with bThrombin may provide a more rapid onset of hemostasis at 3 minutes in PAB procedures. Adverse events were similar between the two thrombins. In patients undergoing vascular surgery, both treatments were similarly well tolerated, although rThrombin demonstrated a superior immunogenicity profile.     

Aggregation of human platelets in plasma by porcine blood cells in vitro is probably mediated by thrombin generation

The infusion of pig progenitor cells into baboons is associated with a thrombotic microangiopathy probably related to the interaction of these cells with the baboon endothelial cells and platelets. We have shown previously that pig peripheral blood mononuclear cells (p-PBMC), are able to activate the human coagulation cascade as they are able to generate thrombin when added to defibrinated plasma. In this work, we have tested the interaction of p-PBMC with human platelets to assess the capacity of p-PBMC to cause platelet aggregation and the possible role of complement activation in this aggregation. Human platelet aggregation assays, using collagen (1 or 2 microg/ml), were performed with platelets in platelet-rich plasma (PRP) or platelets washed by filtration. PRP or washed platelets were also incubated with p-PBMC or human PBMC (h-PBMC) at several concentrations and aggregation was measured. The effect of Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an inhibitor of thrombin, was studied on platelet aggregation caused by the pig cells. Complement activation was measured by deposition of fragment c derived from C3 splitting (C3c) on pig cells incubated with citrated platelet poor plasma (PPP). When human PRP was incubated with p-PBMC, aggregation was a consistent event quantitatively similar to that induced by collagen. No aggregation of washed platelets was observed when these were incubated with p-PBMC or h-PBMC. Aggregation of human platelets in PRP, induced by p-PBMC, was inhibited when DAPA (100 microm) was added to the incubation mixture (23%), indicating that the thrombin inhibitor blocked the capacity of p-PBMC to aggregate human platelets. No deposition of C3c fragments on p-PBMC was detected when the porcine cells were incubated for up to 20 min with citrated PPP. The fact is that p-PBMC induces human platelet aggregation in plasma being thrombin generation a likely explanation for this observation. Our data suggest that, in the system assayed, complement activation is not a cause of platelet aggregation. These findings are relevant for the clarification of the reported thrombotic microangiopathy complicating the intravenous infusion of pig cells in primates in attempts to induce pig tolerance in baboons.     

The effects of thrombin on bovine aortic endothelial and smooth muscle cells

Recent evidence suggests that thrombin interacts with various cell types, stimulating cellular proliferation and protein and prostanoid production. To further delineate its role in vascular healing, we have studied the effects of thrombin on proliferation and matrix production by the cells of the vessel wall. The addition of thrombin (1 unit/ml) to cultures of bovine aortic smooth muscle cells resulted in an increase in cell proliferation (p less than 0.01) and number (p less than 0.03), whereas in cultures of bovine aortic endothelial cells thrombin produced a decrease in cell proliferation (p less than 0.01) and number (p less than 0.02). Thrombin also altered matrix composition in cultures of these cells. In both bovine aortic endothelial cells and bovine aortic smooth muscle cell cultures grown in the presence of thrombin, total protein content was significantly increased when compared to controls (p less than 0.03). In bovine aortic endothelial cell cultures the addition of thrombin resulted in a decrease in collagen content (p less than 0.01) and an increase in sulfated glycosaminoglycan content (p less than 0.02). In contrast, in bovine aortic smooth muscle cell cultures thrombin resulted in an increase in collagen content (p less than 0.03), whereas glycosaminoglycan content was unaffected. These findings suggest that thrombin may significantly influence vascular healing and function by altering cell number and matrix composition.     

Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation

Abstract: The binding of human xenoreactive antibody (XNA) to porcine endothelium with complement (C) activation via the classical pathways is considered the major event triggering hyperacute rejection (HAR) with microvascular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of complement by endothelial cells results in xenogeneic platelet activation in vitro. Methods: Cultured porcine aortic endothelial cells (pEC), human aortic endothelial cells (HAEC) or human umbilical vein endothelial cells (HUVEC) were prepared in suspension (5 × 10⁶/ml) using EDTA-collagenase. Human platelet rich plasma (PRP) and washed platelets with platelet poor plasma (PPP) were prepared from control, drug free, volunteer donors. All aggregation tests used a two-sample, four-channel, model 560 Ca Lumi-Aggregometer (Chronolog Corporation, Havertown, PA). Selected assays for complement (C3a; C5b-C9; CH50; and AP50) were then performed on supernatant fluids. To test the effects of complement inhibition and thrombin antagonists, the following agents were pre-incubated with PRP (or PPP) in various titrations for 10 min at 37°C prior to combination with pEC in the aggregometer: soluble complement receptor typel (sCR1); Cobra Venom Factor (CVF), heparin, hirudin, and anti-CD31 (anti-PECAM). In addition, pEC, HAEC, and HUVEC were incubated with 10–20% human PPP; supernatant fluids were harvested at various time points and used for platelet activation assays and for functional tests of thrombin or levels of thrombin-antithrombin complexes (TAT). Results: pEC but not HAEC/HUVEC resulted in activation of PRP or washed platelets only in the presence of supplemental PPP. Platelet activation could be inhibited by pre-incubation of samples with CVF (2–5 IU/ml to deplete complement components) and to a variable extent with sCRl (1–2 mg/ml). Complement assays confirmed activation of C3 by the classical pathway with reduction of the CH50 while C6 deficient samples also supported platelet activation. Aggregation of platelets was inhibited by preincubation of PRP with concentrations of hirudin (1 unit/ml) and heparin (5–10 units/ml) sufficient to neutralize thrombin generated. Supernatant fluids containing PPP removed from pEC were found to have increased levels of thrombin and thrombin-antithrombin complexes and could also activate platelets in a hirudin sensitive manner. Discussion: Platelet and pEC combinations underwent aggregation only in the presence of complement components. This process appeared independent, at least in part, of the assembly of terminal complement components. Consequent pEC generation of thrombin appeared adequate to trigger platelet aggregation which could in turn be inhibited by hirudin or heparin in vitro.     

Mechanisms and attenuation of hemostatic activation during extracorporeal circulation.

Patients undergoing cardiac surgery with cardiopulmonary bypass are at risk for excessive microvascular bleeding, which often leads to transfusion of allogeneic blood and blood components as well as reexploration in a smaller subset of patients. Excessive bleeding after cardiac surgery is generally related to a combination of several alterations in the hemostatic system pertaining to hemodilution, excessive activation of the hemostatic system, and potentially the use of newer, longer-acting antiplatelet or antithrombotic agents. Although several nonpharmacologic strategies have been proposed, this review summarizes the role of pharmacologic interventions as means to attenuate the alterations in the hemostatic system during CPB in an attempt to reduce excessive bleeding, transfusion, and reexploration. Specifically, agents that inhibit platelets, fibrinolysis, factor Xa and thrombin, as well as broad-spectrum agents, have been investigated with respect to their role in reducing consumption of clotting factors and better preservation of platelet function. Prophylactic administration of agents with antifibrinolytic, anticoagulant, and possibly antiinflammatory properties can decrease blood loss and transfusion. Although aprotinin seems to be the most effective blood conservation agent (which is most likely related to its broad-spectrum nature), agents with isolated antifibrinolytic properties may be as effective in low-risk patients. The ability to reduce blood product transfusions and to decrease operative times and reexploration rates favorably affects patient outcomes, availability of blood products, and overall health care costs.     

等容血液稀释对出血性休克家兔凝血酶生成的影响

目的 探讨等容血液稀释对家兔出血性休克时凝血酶生成的影响.方法 成年雄性新西兰家兔24只,随机均分为羟乙基淀粉组(V组)、琥珀酰明胶组(G组)、复方乳酸钠组(R组)和对照组(C组),V、G和R组分别输注羟乙基淀粉130/0.4、琥珀酰明胶和复方乳酸钠,C组不行血液稀释.血液稀释完成后开腹,切除2cm3肝脏组织后立即缝合腹腔皮肤.10 min后,V、G、R三组开始回输自体血,C组回输复方乳酸钠.记录血液稀释前(T0)、血液稀释后(T1)、切除肝脏组织后10 min(T2)、40 min(即自体血回输完成即刻,T3)、70 min(T4)、130 min(T5)时实验兔的MAP和HR,抽取动脉血样行凝血酶生成试验,记录T0～T5时凝血酶的生成延迟时间、峰值、达峰时间、生成潜力值,并观察肝出血自体血复苏的130mn内家兔的生存时间和失血量.结果 G组家兔在T2时、V组在T1～T5时、R组在T1、T2时MAP明显低于T0时(P＜0.05),T2时G、V和R组MAP明显低于C组(P＜0.05).V组家兔在T1～T5时HR明显慢于T0时(P＜0.05).T3时R组家兔的凝血酶生成延迟时间和T3、T4时凝血酶达峰时间明显长于T0时(P＜0.05),T3时V组的凝血酶峰值明显低于T0时(P＜0.05);T1～T5时V组和R组的凝血酶生成潜力值明显低于T0时(P＜0.05).T4时V组和R组的凝血酶生成潜力值明显低于C组(P＜0.05),T3、T4时G组凝血酶达峰时间明显长于V组(P＜0.05).结论 对等容血液稀释的家兔出血性休克模型,羟乙基淀粉130/0.4对凝血功能的影响较琥珀酰明胶和晶体强.     

Contraceptive steroids influence the hemostatic activation state in healthy men

Hormonal contraception for men requires administration of testosterone and gestagens. The effects of a long-acting testosterone ester and 2 different progestins on hemostatic activation parameters were studied in relation to cardiovascular risk. In phase 1, 7 healthy men aged 28-38 years received a single intramuscular injection of 200 mg norethisterone-enanthate (NET-EN) on Day 0. Plasma samples were obtained on Days 0, 14, 41, and 84. In phase 2, 3 groups of 14 healthy men aged 18-45 years received four injections (every 6 weeks) of 1000 mg testosterone undecanoate (TU), plus daily oral placebo or daily oral levonorgestrel (LNG, 250 microg); or four injections (every 6 weeks) of NET-EN. Treatment lasted 24 weeks. Plasma samples were obtained at weeks 0, 16, 24, and 52. All samples were assayed for levels of coagulation factors VIIc, VIIa, XIIc, and XIIa; prothrombin fragment F1+2 (F1+2); antithrombin; plasmin-alpha(2)-antiplasmin-complex (PAP); and fibrinogen. NET-EN alone led to a depletion of sexual hormones and a marked shift in hemostatic parameters with increasing levels of FXIIc, fibrinogen, antithrombin, and F1+2, whereas FVIIc and FVIIa levels decreased. PAP levels increased significantly. Opposite effects were seen in the TU/placebo group, with a significant down-regulation of fibrinolysis and the hemostatic turnover rate. Testosterone effects were attenuated by additional administration of gestagens. The effect of hormonal male contraception using long-acting testosterone esters with or without gestagens was significantly measurable within the hemostatic system. Down-regulation of the hemostatic system with testosterone alone may indicate an antithrombotic effect, whereas clinical consequences of an additional gestagen compound cannot be derived.