A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, β-thromboglobulin and platelet factor 4 in healthy volunteers

A radioimmunoassay was developed for the platelet α-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml^?1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with β-thromboglobulin (β-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at ?20°C. Normal human plasma contained 105.0 ± 31.0 ng thrombospondin ml^?1 compared with β-TG concentrations of 37.2 ± 10.9 ng ml^?1 and PF4 concentrations of 14.7 ± 10.1 ng ml^?1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0–4°C. Release of thrombospondin during clotting of blood occurred at the same time as that of β-TG and PF4 and resulted in a serum concentration of 17.5 ± 5.5 μg ml^?1. Assay of whole blood gave a platelet thrombospondin content of 89.1 ± 28.3 ng/ 10⁶ platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml^?1, and was unrelated to urine flow. The half-life of thrombospondin $\text{in vivo}$ was about 9 h, much longer than that of either β-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.     

Anticoagulant and lipolytic effects of a low molecular weight heparin fraction

The lipolytic and anticoagulant actions of a 4000 dalton low molecular weight (LMW) heparin were compared with unfractionated mucosal heparin after intravenous and various subcutaneous doses in man. I.v. injection of 100 USP units/kg body weight lipoprotein lipase (LPL) activity, and inhibition of factor Xa decreased with a half life twice as long after LMW heparin compared to normal heparin (p < 0.05). There were no differences in half lives for HTGL activity, thrombin inhibition and on aPTT. The area under the activity time curve (AUC) of LPL and factor Xa was double with LMW heparin (p < 0.05). S.c. administration showed that the AUC of LMW heparin on the factor Xa inhibition was 10 times larger compared to normal heparin. LPL activity was released comparable to normal heparin. The effects on HTGL were three times larger compared to normal heparin. There were no differences in half lives. The data show that in contrast to normal heparin LMW heparin is rapidly and completely absorbed from the subcutaneous depots. The pharmacodynamic data of LPL activity and factor Xa inhibition suggest similar release mechanisms.     

Heparin-induced platelet aggregation: Dose/response relationships for a low molecular weight heparin derivative (pk 10169) and its subfractions

Addition of heparin or heparin derivatives to citrate anticoagulated platelet-rich plasma caused platelet aggregation in a dose-dependent manner. Utilizing heparin, a low molecular weight heparin derivative (PK 10169) and its various subfractions, we determined dose/response relationships for platelet aggregation and found that the ability of these agents to cause platelet aggregation was dependent upon the molecular weight of the individual subfraction used. In comparison to unmodified porcine mucosal heparin, the lower molecular weight derivative (PK 10169) yielded a dose/response curve that was shifted down and to the right, and indicated that this agent was less potent in causing platelet aggregation. In addition, as the molecular weight of PK 10169 subfractions decreased, their dose/response curves were progressively shifted down and to the right. The lowest molecular weight subfraction was essentially without platelet aggregating activity. We also measured the anti IIa and anti Xa activities of these agents and concluded that these activities did not appear to correlate with platelet aggregating activity. Platelet aggregation studies with PK 10169 subfractions of high and low affinity for antithrombin III (AT III) indicated that the platelet aggregating activity of these compounds may not be related to their affinity for AT III, but results were not definitive.     

Laboratory monitoring of thromboprophylaxis with low molecular weight and standard heparin

This study was made to evaluate assays for monitoring of low dose heparin thromboprophylaxis and to evaluate its efficacy in reduction of hypercoagulation. Patients with medical diseases scheduled for routine thromboprophylaxis were subcutaneously treated with either 5.000 anti XaU low molecular weight (LMW) heparin once daily (n=20) or 5.000 IU standard (ST) heparin 3 times daily (n= 19). On days 1,2,3, before, 1 and 4 hours after heparin injection APTT, TCT, anti Xa, Heptest, thrombin-antithrombin complexes (TAT), and D-Dimer levels were measured. In the LMW heparin group, median values of APTT and TCT slightly increased after heparin and the ranges of pre- and postinjection values showed extensive overlap. However, values of anti Xa and Heptest markedly increased, showing complete separation of ranges. In the ST heparin group neither APTT, TCT, anti Xa, nor Heptest were significantly different comparing pre- and postheparin values. Half of the patients in both groups had subclinical hypercoagulation at baseline (TAT>5ng/ml, D-Dimer>200ng/ml). On day 3 of prophylaxis this percentage was not significantly decreased. Moreover, several patients in both groups increased in TAT and D-Dimer. In the LMWheparin group, negative correlations between body weight and 4 h postinjection heparin levels were found (anti Xa R=−0.50, Heptest R=−0.31) and between 1 h postinjection heparin and TAT and D-Dimer levels 3 h later (TAT-anti Xa R=−0.58, TAT-Heptest R=−0.64, D-Dimer-anti Xa R=−0.32, D-Dimer-Heptest R=−0.33). These results show that low dose LMW but not ST heparin therapy can be monitored by the anti Xa test or the Heptest.     

The effect of heparin fragments of different molecular weights on experimental thrombosis and haemostasis

The effect of heparin fragments of different molecular weights has been compared with that of conventional sodium heparin on experimental thrombosis in vivo and ex vivo and experimental haemostasis in vivo. In the first part of the study fragments of different molecular weights were given (4,900, 6,500, 9,500 and 22,200 dalton). All preparations including the control gave a significant prolongation of the haemostatic plug formation time in the rabbit mesenteric microcirculation, and all except the fragment with the lowest molecular weight reduced the frequency of jugular vein thrombosis (induced by a combination of endothelial denudation and stasis). There was a correlation between the XaI activity of the different heparin fragments and frequency of thrombosis. Using an ex vivo method (modification of Chandler's model) a dose dependent lag phase until start of thrombus formation was found. In the second part of the study a dose response investigation was made comparing different doses of a fragment (6,500 dalton) with conventional heparin in the same XaI doses (10, 30 and 60 units/kg). Sodium heparin in the highest dose prolonged the haemostatic plug formation time whereas none of the fragment doses did. The lowest dose both of the fragment and conventional heparin did not reduce the frequency of thrombosis, whereas the two higher doses did. Thus it may be possible to obtain preventive effect on thrombus formation with a heparin fragment.     

Influence of acetylsalicylic acid (1.0 g/day) on platelet survival time, β-thromboglobulin and platelet factor 4 in patients with peripheral arterial occlusive disease

In this study we investigated the influence of acetylsalicylic acid (ASA) 1.0 g/day on 111-In-platelet survival time (PST) and on plasma levels of β-thromboglobulin (β-TG) and platelet factor 4 (PF 4) in 37 patients (median age: 63.4 years) with arteriographically proven peripheral arterial occlusive disease (PAOD) in a chronic stable phase. We found a slight but significant increase of PST during therapy with ASA (weighted mean (WM): 184.3 → 193.2 [median]hours, p < 0.05; multiple hit (MH): 182.4 → 192.8 hours, p<0.005) for the total group of patients. Concerning the influence of risk factors of PAOD on PST during ASA-therapy, there was a significant increase of PST only in the nondiabetics (WM: 180.3 → 204.6 hours p<0.01; MH: 176.8 → 195.3 hours, p<0.01). There was a negative correlation between the baseline values of PST and their increase following ASA therapy (WM: R = - 0.63; p<0.0001; MH: R = - 0.61, p<0.0001). The pretreatment levels of β-TG - but not PF 4 - were significantly (p<0.001) elevated compared to healthy controls. Therapy with ASA caused a significant decrease in the plasma levels of β-TG (median: 30.4 → 26.6 ng/ml, p<0.001) and PF 4 (2.95 2.2 ng/ml, p<0.01).     

High heparin released platelet factor 4 in uncomplicated type 1 diabetes mellitus

The role of platelet activation in diabetic microangio-pathy is still controversial. We evaluated the degree of platelet activation in relation to vessel wall damage in three selected, well matched groups of subjects (10 healthy controls; 20 insulin dependent diabetic patients, 10 without microangiopathy and 10 with microangiopathy). We measured βTG and PF4 plasma levels before and 5, 15 and 90 min after a heparin bolus i.v. (5000 U).

βTG basal levels were increased only in diabetic patients with microangiopathy. Diabetic patients without microangiopathy showed significantly higher levels of heparin released-PF4 (HR-PF4) in comparison with healthy controls. High levels of HR-PF4 seen to be an early marker of in vivo increased platelet activation in uncomplicated diabetes mellitus.     

Pharmacokinetics of a low molecular weight heparin, Logiparin, after intravenous and subcutaneous administration to healthy volunteers

In six healthy volunteers we have estimated the pharmacokinetic parameters of the anti factor Xa (AXa) and anti factor IIa (AIIa) activities of a LMW heparin, Logiparin. For the AXa the following parameters were estimated in a 1-compartment model (mean and 95% confidence limits in brackets): elimination half life 82 minutes (60–127 min), absorption half life (s.c.inj.) 200 minutes (137–368 min), bioavailability 90% (24–156 %), and apparent volume of distribution 3.9 1 (3.1–5.2 1). The plasma activity was linearly correlated to the dose given and to the body weight of the volunteer. For the AIIa the parameters estimated in a 1-compartment model were: elimination half life 71 minutes (52–115 min), absorption half life 257 minutes (133–3442 min), bioavailability 67% (44–90 %), and apparent volume of distribution 10.1 1 (7.2–16.7 1). The plasma activity was dependent on dose and body weight but it also seemed to be influenced by individual factors. This study shows that the absorption rate is the rate limiting factor and the explanation for the long lasting effect of this LMW heparin after subcutaneous injection. The slow absorption rate and the high bioavailability are probably the major advantages of LMW heparins compared to conventional heparin.     

Tissue factor pathway inhibitor (TFPI) activity in uremic patients during hemodialysis

We studied tissue factor pathway inhibitor (TFPI) activity during hemodialysis in 10 uremic patients who were not receiving anticoagulant for at least 120 minutes. TFPI activity before dialysis was normal (patients 107 ± 5.8%, controls 104 ± 4.5%). During extracorporeal circuit it rose progressively with a statistically significant difference, reaching a plateau between 60 and 120 minutes. Since thrombin induces a marked redistribution and release of TFPI from stimulated endothelial cells and platelets contain about 10% of TFPI activity that is secreted following activation it is possible that thrombin-induced release of TFPI by endothelium and platelets could account for the increased TFPI we found during hemodialysis. To investigate this possibility we measured during dialysis β-thromboglobulin (β-TG), thrombinantithrombin complex (TAT) and prothrombin fragment 1.2 (F 1.2). The increased levels of β-TG, TAT and F1.2 we noted during extracorporeal circuit are in keeping with this concept.

One hundred eighty minutes after initiation of dialysis, by which time all patients were receiving heparin there was a further increase in TFPI (to more than 200% of baseline), due to the presence of the glycosaminoglycan. This was due the previously reported displacement by heparin of the major intravascular pool of TFPI, from endothelial cell surfaces.     

低分子肝素联合丹参注射液治疗急性脑梗死的效果分析

目的探讨低分子肝素和丹参注射液联合治疗急性脑梗死的临床效果。方法选取2012-12—2015-01我院诊治的急性脑梗死患者95例,随机分为研究组(n=48例)和对照组(n=47例),对照组实施基础治疗与丹参注射液,观察组加用低分子肝素治疗。比较2组治疗前后血液流变情况、神经功能改变情况(NIHSS评分),并评定临床疗效、并发症情况。结果2组治疗后血液流变学指标(纤维蛋白原、红细胞比积、血浆黏度、全血低切黏度、全血高切黏度)均明显低于治疗前(P0.05),且研究组较对照组降低更明显(P0.05);治疗后7、14、28d2组NIHSS评分均较治疗前明显降低(P0.05),且研究组各时点NIHSS评分较对照组更低(P0.05);研究组与对照组总有效率分别为91.67%和74.47%,研究组高于对照组(P0.05);2组并发症发生情况无明显差异(P0.05)。结论低分子肝素联合丹参注射液治疗急性脑梗死,患者脑部循环明显改善,效果显著,值得推广。     

Sulfated low molecular weight lignins,allosteric inhibitors of coagulation proteinases via the heparin binding site,significantly alter the active site of thrombin and factor xa compared to heparin

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold.     

Discordance between the anti-Xa activity and the antithrombotic activity of an ultra-low molecular weight heparin fraction

The relationship between the in vivo antithrombotic effect of heparin and ex vivo anti-Xa activity has been investigated using an animal thrombosis model. Three low molecular weight heparins were compared with the standard heparin from which they were fractionated. All four heparins showed a dose-dependent antithrombotic effect enabling the relative antithrombotic and anti-Xa activities to be compared over a dosage range. A correlation between ex vivo anti-Xa heparin levels and antithrombotic effect was demonstrated for the standard (MW 16,000), intermediate (MW 7,600) and low (MW 4,600) molecular weight heparins but not for the ultra-low molecular weight (MW 3,000) fraction. The lack of relationship between anti-Xa activity and inhibition of thrombosis for the very low molecular weight fraction indicates that a very high anti-Xa activity (measured in vitro or ex vivo) is not always predictive of in vivo antithrombotic efficacy. These findings suggest that other properties of low molecular weight heparins contribute to their antithrombotic effectiveness.     

The extrinsic fibrinolytic system in survivors of myocardial infarction

The extrinsic fibrinolytic system was assessed among 124 consecutive survivors of acute myocardial infarction below 70 years of age. In samples drawn 3 months after discharge from hospital, the PAI-1 levels were higher and the tPA activities were lower than among elderly healthy controls. In contrast, the AMI survivors had higher tPA antigen levels at rest and after venous occlusion, and higher tPA activities after venous occlusion. Among patients having PAI-1 levels >10 IU/ml, there was a positive correlation between PAI-1 and serum triglycerides, and a negative correlation between PAI-1 and age; this group was also significantly younger than the subgroup having <-10 IU/ml of PAI-1.

There were thus multiple disturbances of the extrinsic fibrinolytic system among these patients. As cardiovascular risk factors, these disturbances appear to be relatively more important the younger the patients are.     

Prevention of experimental venous thrombosis in rabbits with low molecular weight heparin, dextran and their combinations, administered before or during induction of venous endothelial trauma

An in vivo experimental venous thrombosis model based on endothelial damage and flow reduction was used to investigate the effect of low molecular weight heparin (LMWH) alone and in combination with dextran and the effect of surgical and endothelial trauma on thrombus formation, formation of occlusive thrombi and thrombus weights. Five groups with 15 rabbits in each were studied. Two groups received dalteparin (50 anti-Xa IU/kg i.v.) before surgical trauma or after, during the endothelial trauma and two groups received dalteparin (50 anti-Xa IU/kg i.v.) with dextran 70 (1 g/kg i.v.) before surgical trauma or after, during the endothelial trauma. Compared to a control group (saline) all treatment regimes reduced significantly the frequency of thrombosis and occlusive thrombi as well as thrombus weights. No significant difference was observed between the identical treatment groups when the substances were introduced before respective after surgical trauma. It is concluded from the present study that thromboprophylaxis with LMWH in this particular in vivo model, given before or after surgical trauma is equally effective. Dextran has a certain augmenting thromboprophylactic effect when added to LMWH in this model.     

Effect of EDTA and PGE1 on beta-thromboglobulin liberation from platelets

The aim of this study was to evaluate the effect of PGE₁ and EDTA on liberation of β-thromboglobulin (βTG) from platelets in vitro. Liberation of BTG was followed in citrated blood at room temperature for 120 minutes after venesection. PGE₁ reduced βTG liberation, and maximal inhibition was attained by concentrations greater than 2 × 10⁻⁶M. EDTA induced the efflux of βTG. This EDTA-induced efflux was delayed but not prevented by PGE₁ and by citrate; it was not found at 0–4°C. Therefore the use of EDTA to prevent βTG liberation during sampling for in vitro or in vivo studies depends heavily on modifying factors such as PGE₁ and low temperature, and on the time taken to process samples. Its effectiveness must be in some doubt where the platelets may be sufficiently stimulated to overcome these modifying influences, or where handling of samples is less than optimal.     

拜阿司匹灵对脑梗死患者血浆血小板4因子的影响

目的观察脑梗死患者用拜阿司匹灵治疗前、后血浆血小板4因子(PF4)的变化。方法70例脑梗死患者被随机分为不用拜阿司匹灵组(n=40)和服用拜阿司匹灵组(100mg/次,睡前服用,n=30),其他治疗两组间无差异。用酶标免疫测定法测定二组治疗前、治疗后7d及30例正常对照组血浆PF4的含量。结果急性脑梗死不用拜阿司匹灵组的患者血浆PF4治疗前为15.12±6.32ng/ml,治疗后为9.83±4.93ng/ml,治疗后PF4的明显下降(P<0.01),但显著高于正常对照组(6.50±2.70ng/ml,P<0.01);服用拜阿司匹灵组的患者血浆PF4治疗前为15.48±3.16ng/ml,治疗7d后为7.66±1.47ng/ml,较治疗前明显下降(P<0.01),也明显低于不用拜阿司匹灵组的患者(P<0.01),但仍高于正常对照组(6.50±2.70ng/ml,P<0.05)。结论拜阿司匹灵能降低脑梗死患者血浆PF4含量水平,抑制血小板活性。     

Inhibition of the availability of platelet factor 3 by increased concentration of lecithin in platelets caused by gonadal steroids

Platelet factor 3 (PF-3) activity and the phosphatide composition of plasma and platelets were studied in a group of oophorectomized women on estrogen-gestagen treatment. Based on the results the following hypothesis is put forward. An increased concentration of lecithin in the platelets stabilizes the platelet membrane and inhibits the conformational changes of the membrane which is supposed to precede the “availability” of PF-3. Estrogen treatment induces a decrease of plasma lysolecithin resulting in a decreased formation of platelet lecithin. The increased PF-3 activity observed during the estrogen dominated phase may thus not be due to an increase of lipids promoting clotting but to a decrease of platelet lecithin which normally prevents the “release” of clot promoting activities.