H.D. Bruhn

Anticoagulant fucoidan fractions from Fucus vesiculosus induce platelet activation in vitro

Anticoagulant fucoidan fractions of different molecular weight and sulfate content were prepared and investigated for their effects on platelet function in vitro. The fucoidan fractions were incubated with human platelet rich plasma (PRP) at concentrations of 5, 10 and 50 micrograms/ml. Platelet activation was subsequently studied by a standard aggregation assay and flow cytometric determination of the activation dependent platelet-surface markers CD62p (P-selectin, GMP-140) and CD63 (GP53). All fucoidan fractions induced irreversible platelet aggregation in a dose-dependent manner. Comparing fractions of identical molecular weight (100 kDa) the low sulfate content fucoidan FF5 (S = 7.6%) exerted a significantly greater effect than the highly sulfated fucoidan FF7 (S = 10.2%) over the whole concentration range (n = 5, P < 0.05). Among fractions of identical sulfate content fucoidan-induced platelet aggregation was also found to depend on the molecular weight of the fucoidan. At concentrations of 10 and 50 micrograms/ml the high molecular weight fraction FF7/1 (150 kDa) showed a significantly greater effect than the 50 kDa fraction FF7/3 (24.8 +/- 6.7 vs. 7.0 +/- 3.5 and 54.6 +/- 13.5 vs. 15.0 +/- 9.0%, respectively; mean +/- SD, n = 5, P < 0.05). The molecular weight dependence of the fucoidan effect was also reflected by the flow cytometric data. Coincubation of FF7/1 and FF7/3 (10 micrograms/ml) with PRP increased the number of CD62p and CD63 positive platelets by 9.0 +/- 3.3 vs. 2 +/- 1.9 and 7.1 +/- 2.4 vs. 3.2 +/- 2.6% over control values, respectively (n = 5, P < 0.05). In conclusion, our results show that the low molecular weight fucoidan FF7/3 combines potent anticoagulant and fibrinolytic properties with only minor platelet activating effects and is therefore a suitable substance for further pharmacological studies.

Investigations of Coagulation System and Fibrinolysis in Patients with Disseminated Adenocarcinomas and Non-Hodgkin’s Lymphomas

Indicating activation of coagulation fibrinopeptide A (FPA) was elevated in 80.1% (mean = 10.5 ng/ml; P less than 0.01) and thrombin-antithrombin III complexes in 58.3% (TAT; mean = 5.3 ng/ml; p less than 0.05) in patients with adenocarcinomas (n = 57). In patients with non-Hodgkins lymphomas (n = 30), however, elevation was observed only in 66.6% (FPA) and in 42.8% (TAT). Incidence of thrombosis is high only in the first group Local fibrinolysis explains elevated D-dimer in adenocarcinomas (1,818 ng/ml; p less than 0.01) and in non-Hodgkins lymphomas (576 ng/ml; p less than 0.05). Significantly increased t-PA antigen was not committed by adequately increased t-PA activity in adenocarcinomas, because of high levels of the acute-phase protein, plasminogen activator inhibitor (mean = 25.3; p less than 0.01), indicating systemic hypofibrinolysis. Hemostatic disorder in patients with malignancy can be attributed to a combination of acute-phase reaction and an activation of coagulation.

Influence of heparin treatment on biochemical markers of an activation of the coagulation system.

Monitoring of anticoagulant treatment is not yet satisfactory. Otherwise optimal treatment control in special situations as low dose heparin prophylaxis in hip surgery or high dose anticoagulant treatment in patients after coronary stent implantation may be desirable. Recently available thrombin markers were analyzed in 51 patients under low dose (group 1) and 30 patients under high dose therapy with unfractionated heparin (group 2a and b) as well as in controls (n = 26). Before therapy these parameters were significantly elevated in both patient groups. Elevated thrombin-antithrombin III-complexes (TAT) despite adequate prolongation of aPTT under high dose heparin in 38.2% of patients indicate that therapeutic concentrations of heparin in these cases are insufficient for depressing this parameter completely. During low dose therapy only prothrombin fragment (F1 + 2) significantly decreased. This may be explained by catalytic induction of TAT-complex formation by heparin. Decrease of D-Dimer under heparin therapy in both groups does not parallel with TAT and F1 + 2 but was more prolonged. This can be explained by dependence of the D-Dimer level on spontaneous fibrinolytic activity and by a longer plasma half-life as well as a chronic and continuous fibrinolytic process in an older thrombus. In conclusion, thrombin markers seem to be helpful in estimating anticoagulant treatment efficacy. As a consequence, anticoagulant treatment has to be intensified in high-risk patients for complete depression of these markers. Whether the benefit of higher heparin doses is worth the risk of drug-induced hemorrhage, however, remains to be clarified in clinical studies.

Does protamine chloride neutralize low molecular weight heparin sufficiently

The heparin neutralizing properties of protamine chloride on conventional heparin (porcine mucosa) and on low molecular weight heparin (Kabi 2165) were studied in vitro. Protamine chloride neutralized 99% of the delaying effect of conventional heparin on the activated partial thromboplastin time, whereas only 70% of the effect of low molecular weight heparin was neutralized. The neutralizing effect of protamine chloride on the inhibition of factor Xa (clot test) was 95% for conventional heparin and 55% for low molecular weight heparin, whereas the effect of both heparin preparations on the thrombin inhibition could be completely neutralized. We conclude that conventional heparin is neutralized more effectively in vitro by protamine chloride than is the low molecular weight heparin. The findings do not exclude that protamine chloride is able to suppress in vivo bleedings caused by low molecular weight heparin.

Immunological and functional determination of the protease inhibitors, protein C and antithrombin III, in liver cirrhosis and in neoplasia

Using a new rapid coagulant method, protein C activity (PC act) was determined in liver cirrhosis and malignancies and compared with PC antigen and AT III values. PC was decreased in a more pronounced manner than AT III in liver cirrhosis, mainly due to impaired synthesis. This is of special clinical interest because PC proved to be a high sensible indicator of liver cell dysfunction. Decreased levels of PC act (PC ratio act/ag less than 1) in decompensated liver cirrhosis may be caused by the synthesis of dysfunctional PC and/or vitamin K deficiency with production of undercarboxylated PC most sensitively registered by this coagulant assay. An increased clearance of in vivo activated PC induced by DIC may play an insignificant role. In patients with liver metastases, PC act (but not AT III and immunological parameters) was significantly reduced, supporting the conclusion that in these patients liver dysfunction concomitant with synthesis of dysfunctional PC must be discussed as the main cause of this alteration.

Swimming and hemostasis during rehabilitation in patients with coronary heart disease

Coronary heart disease and acute myocardial infarction are frequent causes of death in Western industrial countries. Therefore, prevention of myocardial infarction and rehabilitation after myocardial infarction are of significant medical interest. In epidemiologic studies, physical inactivity was detected as a coronary risk factor and, on the other hand, a meta analysis of 22 randomized studies showed that continuous physical exercise induces a reduction of cardiac mortality by 20% [25,26]. However, the risk of hypercoagulability induced by exercise exists especially among patients with heart and vessel diseases [11]. The influence of physical exercise by running and bicycle ergometer on the human haemostatic system has been well investigated [6,11,14,33]. However, for swimming there are nearly no data available, although swimming is included in the training program of many of the so-called coronary sport groups. As an endurance sport with training effect swimming is suited as rehabilitation. This is only true for patients with normal physical workload, since everybody has an increased energy turnover equal to an ergometer workload of 75 W in water at 25–29 jC even without physical activity [22]. Thus, knowledge about the activation of clotting and the fibrinolytic system is important. It has been demonstrated that patients after physical exercise by running and cycling have an activation of clotting factors with the potential risk for thrombosis [6,11,14,33]. Comparable investigations for swimming are lacking. Therefore, in the present study parameters for the assessment of clotting and fibrinolytic system in patients before and after swimming were investigated.

Influences of thrombin, factor XIII and fibronectin on the growth of tumor cells and leukemic cells in vitro

SummaryThrombin, factor XIII and fobroncctin were incubated with cultures of mouse sarcoma cells, human cervix carcinoma cells (HeLa cells) and cells of an acute lymphoblastic leukemia. Thrombin induced a significant increase of3H-thymidine uptake into cells with a 1,5- to 2-fold increase of cell count. The cells of an acute lymphoblastic leukemia showed a similar response to the influence of thrombin. Factor XIII merely induced an increase of3H-thymidine uptake in tumor cells, the cell count remained constant. These results differ from experiments with cultures of normal fibroblasts. Decreased receptor density in tumor cells toward factor XIII and thrombin would play a role so that the mitogenic stimulus is impaired. The cells of an acute lymphoblastic leukemia showed under the influence of factor XIII a significant increase of cell count and thymidine uptake. HeLa cell growth was optimal at low fibronectin concentrations. Fibronectin concentrations of 1 mg/ml to 3 mg/ml inhibited HeLa- and mouse sarcoma cell growth.

Thrombin, Faktor XIII und Fibronectin als Regulatoren der Proliferation von Tumorzellen und Gefäßwandzellen

In der Gewebekultur werden Fibroblasten, Endothelzellen und glatte Muskelzellen aus Arterienwand von Thrombin und Faktor XIII in ihrem Wachstum stimuliert, wahrend Fibronectin die Proliferation dieser Zellen hemmt. Vergleichende Untersuchungen mit Tumorzellen zeigen eine stimulierende Wirkung von Thrombin und Faktor XIII auf Sarkomzellen und HeLa-Zellen. Fibronectin hemmt das Wachstum von Sarkomzellen. Die Proliferation von HeLa-Zellen wird dagegen von Fibronectin in physiologischen Konzentrationen deutlich stimuliert. Es werden mogliche Wirkmechanismen der Wachstumsfaktoren diskutiert und Bezuge zu in vivo-Phanomenen hergestellt.

Die immunologische und funktionelle Protein C-Bestimmung bei verschiedenen internistischen Erkrankungen

SummaryA new practicable and precise functional protein C evaluation test is based on the activation of protein C by a snake venom activator and determination of PC activity by its property to prolong the aPTT in a clotting assay (VK=1.9% and 4% for intra- and interassay variance respectively). In 40 healthy controls there was a good correlation (r=0.74) between the functional and immunological (ELISA) evaluation. In 123 patients with both evaluation methods but more pronounced with the measurement of the protein C activity a significant protein C deficiency was found in the patient groups with disseminated solid tumors, inflammatory diseases and myocardial infarction. Besides detection of hereditary PC deficiency Type II (generation of functionally abnormal PC) the functional assay profits by recording PC inhibitor complexes and otherwise dysfunctional PC in DIC. Thus, in patients with hematological neoplasias, only protein C activity was significantly decreases. Decrease of PC activity was more pronounced compared to PC Ag in liver disease indicating synthesis of functionally deficient PC, and in oral anticoagulant treatment due to detection of PIVKA-PC.A new practicable and precise functional protein C evaluation test is based on the activation of protein C by a snake venom activator and determination of PC activity by its property to prolong the aPTT in a clotting assay (VK = 1.9% and 4% for intra- and interassay variance respectively). In 40 healthy controls there was a good correlation (r = 0.74) between the functional and immunological (ELISA) evaluation. In 123 patients with both evaluation methods but more pronounced with the measurement of the protein C activity a significant protein C deficiency was found in the patient groups with disseminated solid tumors, inflammatory diseases and myocardial infarction. Besides detection of hereditary PC deficiency Type II (generation of functionally abnormal PC) the functional assay profits by recording PC inhibitor complexes and otherwise dysfunctional PC in DIC. Thus, in patients with hematological neoplasias, only protein C activity was significantly decreases. Decrease of PC activity was more pronounced compared to PC Ag in liver disease indicating synthesis of functionally deficient PC, and in oral anticoagulant treatment due to detection of PIVKA-PC.

Gerinnungsstörungen bei Tumoren und Hämoblastosen

Zur naheren Charakterisierung der Gerinnungsstorungen, die nicht selten im Verlauf maligner Erkrankungen zu thromboembolischen oder hamorrhagischen Komplikationen fuhren, wurden Gerinnungsanalysen bei