Studies on a complex mechanism for the activation of plasminogen by kaolin and by chloroform: the participation of Hageman factor and additional cofactors.

As demonstrated by others, fibrinolytic activity was generated in diluted, acidified normal plasma exposed to kaolin, a process requiring Hageman factor (Factor XII). Generation was impaired by adsorbing plasma with glass or similar agents under conditions which did not deplete its content of Hageman factor or plasminogen. The defect could be repaired by addition of a noneuglobulin fraction of plasma or an agent or agents eluted from diatomaceous earth which had been exposed to normal plasma. The restorative agent, tentatively called Hageman factor-cofactor, was partially purified by chromatography and had an apparent molecular weight of approximately 165,000. It could be distinguished from plasma thromboplastin antecedent (Factor XI) and plasma kallikrein, other substrates of Hageman factor, and from the streptokinase-activated pro-activator of plasminogen. Evidence is presented that an additional component may be needed for the generation of fibrinolytic activity in mixtures containing Hageman factor, HF-cofactor, and plasminogen.The long-recognized generation of plasmin activity in chloroform-treated euglobulin fractions of plasma was found to be dependent upon the presence of Hageman factor. Whether chloroform activation of plasminogen requires Hageman factor-cofactor was not determined, but glass-adsorbed plasma, containing Hageman factor and plasminogen, did not generate appreciable fibrinolytic or caseinolytic activity. These studies emphasize the complex nature of the mechanisms which lead to the generation of plasmin in human plasma.

Bronchial carcinoma in von Willebrand's disease. Successful removal after hemostasis with lyophilized cryoprecipitate.

A man with severe von Willebrand's disease who had hemoptysis was followed-up for 2 1/2 years for continued hemoptysis and was discovered to have a bronchial carcinoma. Under close hematological surveillance, an uncomplicated pneumonectomy was performed, using a new factor VIII product--freeze-dried cryoprecipitate. This case illustrates that hemoptysis in von Willebrand's disease cannot always be attributed to the disease itself: that continued hemoptysis requires regular bronchoscopy and that major surgery can be performed in von Willebrand's disease with effective correction of the coagulation defect using freeze-dried cryoprecipitate.

Effect of dipyridamole alone and in combination with aspirin on whole blood platelet aggregation, PGI2 generation, and red cell deformability ex vivo in man.

STUDY OBJECTIVE: The aim was to investigate the effects of dipyridamole, aspirin, and a combination of dipyridamole plus aspirin on platelet aggregation in whole blood, PGI2 generation, and red cell deformability ex vivo. SUBJECTS: were 16 male volunteers, aged 22-39 years, mean age, 26.6 years. DESIGN: This was a randomised, double blind, placebo controlled trial. The volunteer received each of the following treatments 10 days apart: dipyridamole 200 mg; aspirin 300 mg; dipyridamole 200 mg plus aspirin 300 mg; matched placebos. MEASUREMENTS AND MAIN RESULTS: Blood was taken for platelet function tests, PGI2 metabolite assay, and red cell deformability before and 2 h after the trial dose was taken. Platelet aggregation was quantified by measuring the fall in single platelet count after stimulation with 2 micrograms.ml-1 collagen or 50 nM platelet activating factor (PAF), or by rollermixing aliquots of blood to initiate spontaneous aggregation. The platelet function tests were completed at 37 degrees C within 10 min of venepuncture. The stable metabolite of PGI2, 6-keto PGF1 alpha, was measured in serum. There was inhibition of spontaneous platelet aggregation by dipyridamole (p less than 0.004), aspirin (p less than 0.005), and the combination of dipyridamole plus aspirin (p less than 0.0001) as compared with placebo. PAF induced platelet aggregation was inhibited by dipyridamole (p less than 0.002) and the combination of dipyridamole plus aspirin (p less than 0.0001) but aspirin alone had no inhibitory effect. Collagen induced platelet aggregation was inhibited by all three treatments: dipyridamole (p less than 0.06), aspirin (p less than 0.0001), and the combination of dipyridamole plus aspirin (p less than 0.0001). PGI2 generation was markedly inhibited by aspirin (p less than 0.0001) and the combination doses (p less than 0.0001) but was unaffected by dipyridamole alone. Of the three active treatments, only dipyridamole alone significantly (p less than 0.001) increased red cell deformability; there was a modest decrease in red cell deformability with aspirin. CONCLUSIONS: The results with PAF support the view that dipyridamole inhibits platelet activation by more than one mechanism; the effect on collagen induced and spontaneous platelet aggregation suggests that the effect of the combination doses is additive and that on red cell deformability the synergy is negative.

Platelet aggregation inhibitory effects of the new positive inotropic agents pimobendan and UD CG 212 in whole blood.

A method is described for studying platelet function in human whole blood immediately after venepuncture in order to evaluate the antithrombotic potential of new pharmacological agents. In this method, platelet aggregation is quantified by measuring the fall in single platelet count, by using a whole blood platelet counter. We have investigated the platelet aggregation inhibitory effects of the new positive inotropic agents pimobendan and UD CG 212 (reported to be Ca++ sensitisers and phosphodiesterase inhibitors), alone and in combination with dipyridamole. Venous blood was drawn directly into prewarmed (37 degrees C) plastic syringes containing anticoagulants (3.2% trisodium citrate solution) plus a platelet aggregation inhibitor. Spontaneous platelet aggregation (SPA) was studied by roller mixing aliquots of blood in the collecting syringes for 6 min at 37 degrees C. Collagen induced platelet aggregation was studied by incubating aliquots of blood with 1 microgram/ml collagen on a shaking water bath for 3 min. In the absence of an inhibitor, there was a 50% fall in single platelet count due to SPA and a 65% fall was induced by collagen. Both SPA and collagen induced aggregation responses were inhibited by pimobendan (0.5-10 microM) and UD CG 212 (0.5-10 microM), in a dose dependent manner. A combination of 10 microM dipyridamole with 2 microM pimobendan or UD CG 212 was markedly a more effective inhibitor of platelet aggregation than a high dose of either inhibitor alone. It is suggested that the present method is simple and rapid, with minimal sample processing, and therefore the results may be protected from serious artifacts.(ABSTRACT TRUNCATED AT 250 WORDS)

Free radical pathology in chronic arterial disease.

The generation of toxic oxygen metabolites is more usually associated with inflammation. However, pathological free radical reactions can cause tissue damage by adversely affecting prostacyclin (PGI2) synthesis allowing initiation of coagulation. We have assessed changes in the red cell defence to toxic oxygen metabolite generation, viz measurement of glutathione concentration (GSH) and superoxide dismutase activity (SOD). GSH and SOD were measured in 20 patients with peripheral arterial disease, 22 patients with vasculitis, and 11 patients with angina, and compared to 17 matched controls. The 53 subjects with arterial disease had significantly lower SOD levels: in contrast GSH levels were significantly higher. Extracellularly plasma thiol levels (PSH) were low and caeruloplasmin (Cp) levels were high. We suggest that free radical pathology exists not only in inflammatory vascular disease but also in atherosclerosis.

Dipyridamole inhibits red cell-induced platelet activation.

We have recently shown that red blood cells can induce spontaneous platelet aggregation (SPA) in whole blood ex vivo, which could be inhibited by dipyridamole. Since this drug, at therapeutic doses is not an effective inhibitor of platelet aggregation in platelet rich plasma, the inhibition of platelet interaction with the red cell was thought to be the mechanism of its action. Values for the percentage fall in the single platelet count due to SPA in whole blood after 3 and 6 min rollermixing were: control 15 +/- 2.2 and 42 +/- 2.9; 6 microM dipyridamole 6 +/- 1.1 (P less than 0.001) and 31 +/- 2.6 (P less than 0.01); 12 microM dipyridamole 2 +/- 0.9 (P less than 0.0005) and 22 +/- 2.3 (P less than 0.0005) (mean +/- SEM, n = 10). Electron microscopic observation revealed that the aggregation involves an initial platelet adhesion to the red blood cell; the adherent platelets then become activated and serve as foci for the growing aggregates. Dipyridamole appeared to inhibit the initial platelet adhesion to the red cell (the principal trigger mechanism for SPA) which may mimic the initiation of thrombosis in some situations in vivo. The inhibitory effect of dipyridamole on the platelet-red cell interaction suggests that this drug has antithrombotic potential in situations where red blood cells have a trigger role in platelet activation and may explain why the drug has been more effective in some situations than in others.

Red blood cells mediate spontaneous aggregation of platelets in whole blood.

The influence of red blood cells on spontaneous platelet aggregation (SPA) has been studied ex vivo. Platelet aggregation was quantified by measuring the fall in single platelet count using a new whole blood platelet counter. When aliquots of whole blood and autologous platelet rich plasma (PRP) were roller-mixed at 37 degrees C, a marked fall in platelet count occurred in whole blood due to SPA but platelet count remained almost unchanged in PRP. When blood from healthy young controls, aged 20-35 years, was compared with healthy old controls, aged 48-80 years, and patients with thrombotic complications, the extent of SPA was in the order: thrombotic patients greater than old controls greater than young controls. Prostacyclin and the new stable prostacyclin analogue Iloprost, at 8 nM effectively inhibited SPA. 2-Chloroadenosine (10 microM) which is an inhibitor of ADP-induced platelet aggregation was also an effective inhibitor of SPA. Acetylsalicylic acid (56 microM) and the thromboxane A2 receptor blocker BM13.177 (0.5 microM) only partially inhibited SPA. ADP from red blood cells is suspected to mediate red cell-induced SPA. However, the possibility that the red cells have an important physical role in SPA cannot be ruled out.

B cell dysfunction in haemophilia in the absence and presence of HIV-1 infection.

56 haemophiliacs selected on the basis of HIV-1 antibody status, liver disease grade and mean annual dose of clotting factor concentrate used were studied. Spontaneous and stimulated IgG and IgM production in vitro were measured. HIV-1 infection was associated with increased spontaneous immunoglobulin production and an impaired response to pokeweed mitogen and Staph Aureus protein A. Implying a shift in the proportions of partially and fully activated B cells. In the absence of HIV-1 infection there was a shift to a greater proportion of partially activated B cells in patients with severe liver disease. The remainder had in vitro immunoglobulin production comparable to controls. B cell abnormalities occur early in the course of HIV-1 infection. Liver disease and not clotting factor concentrate treatment cause B cell abnormalities in the absence of HIV-1 infection in haemophilia.

Effect of dipyridamole on spontaneous platelet aggregation in whole blood decreases with the time after venepuncture: evidence for the role of ADP.

Spontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40, and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37 degrees C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 microM at each time interval; (b) 0.5-100 microM at 3 and 30 minutes and (c) 15 microM in combination with 100 microM adenosine, 8 microM 2-chloroadenosine (2Clad, an ADP receptor blocker) and 50 microM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 microM Dipy and 10 microM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 microM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

Haemostatic effects of ketorolac with and without concomitant heparin in normal volunteers.

Ketorolac is a potent cyclo-oxygenase inhibitor used for the treatment of postoperative pain. It is known to have anti-platelet properties. The aim of this study was to determine the effect of ketorolac on haemostasis both alone and in combination with low dose heparin in 12 healthy male volunteers. Each volunteer received the following drug combinations in a double blind, placebo controlled, cross over manner: ketorolac placebo/heparin placebo, ketorolac active/heparin placebo, ketorolac active/heparin active and ketorolac placebo/heparin active. Ketorolac significantly prolonged bleeding time, inhibited platelet aggregation to arachidonic acid and collagen and platelet thromboxane production. Heparin had no effect on bleeding time or platelet function, but significantly prolonged the kaolin cephalin clotting time and increased anti-Xa levels. Ketorolac had no effect on the kaolin cephalin clotting time or anti-Xa levels and no interaction was found between ketorolac and heparin in any of the investigations. The prolongation of bleeding time seen with ketorolac is unlikely, to be of any major clinical significance as almost all subjects remained within the normal range; however, it should be used with caution in subjects with haemostatic problems.

Blood viscosity and haemostasis in the nephrotic syndrome.

Blood viscosity and its major determinants (haematocrit, plasma viscosity and fibrinogen) as well as several haemostatic variables were measured in 21 patients with the nephrotic syndrome, and 21 controls matched for age, sex, smoking habit and serum creatinine. Blood viscosity was significantly increased in the nephrotic group, measured at a low shear rate (mean increase 41%, p less than 0.01) and at a high shear rate (mean increase 25%, p less than 0.01). Haematocrit was not significantly increased, but plasma viscosity was significantly higher (p less than 0.01), associated with increased plasma macroglobulins especially fibrinogen, which was increased to double the plasma concentration of the control group (p less than 0.01). Nephrotic subjects also had increased plasma levels of alpha 2-macroglobulin, factor VIII activity, factor VIII antigen and beta-thromboglobulin; differences in antithrombin III, fibrin degradation products, plasminogen, and platelet count were not significant. We suggest that increased blood and plasma viscosity may play a role in the vascular complications of the nephrotic syndrome.

Plasma beta-thromboglobulin, fibrinopeptide A and B beta 15-42 antigen in relation to postoperative DVT, malignancy and stanozolol treatment.

Plasma levels of betathromboglobulin (BTG), fibrinopeptide A (FPA) and B beta 15-42 fragment, indices of platelet release, thrombin generation and plasmin activity respectively, were measured in 32 high risk patients during a double blind study of a single dose of the anabolic steroid stanozolol (50 mg IM) in the prevention of DVT after major gastro-intestinal surgery. The prevalence of malignancy and the incidence of DVT (125I fibrinogen scan) were similar in the two treatment groups. On the first postoperative day, BTG, FPA and B beta 15-42 levels were increased in most patients. Plasma BTG levels were significantly increased on the first post-operative day in patients who developed a DVT (n = 14) compared to those patients who did not (n = 18). A significant increase in FPA levels was found in the DVT group, 7 days after surgery. On the morning before surgery, plasma B beta 15-42 levels were significantly increased in patients who developed a DVT. In patients undergoing surgery for early malignancy (n = 17), we observed a pre-operative increase in FPA levels when compared to patients without malignancy. At post-operative day 7, B beta 15-42 levels were significantly increased in patients who received stanozolol (n = 15), when compared to the placebo group, suggesting that intramuscular stanozolol increases fibrinolysis in vivo.

The effects of acute smoking on platelet behaviour, fibrinolysis and haemorheology in habitual smokers.

There is an increased frequency of arterial thrombosis in cigarette smokers. The changes in blood coagulation seen in these subjects have been studied by many workers but results have not always been in agreement. We wished to study the effects of acute smoking on platelet behaviour, fibrinolysis and haemorheology in ten habitual smokers, and to compare these results with non-smoking controls. Results show that the smoking group had higher plasma fibrinogen (p less than 0.04), lower plasminogen (p less than 0.02) and plasminogen activator (p less than 0.05), and higher plasma viscosity (p less than 0.003). The changes seen in cigarette smokers after smoking three cigarettes were an increase in the rate of platelet aggregation to ADP (p less than 0.02), an increase in alpha 2M, (p less than 0.02), and factor VIII RAG (p less than 0.05). Plasma viscosity was decreased (p less than 0.02) as was red cell deformability (p less than 0.02). We confirm an increased tendency to hypercoagulability in smokers compared to controls which becomes more pronounced immediately after smoking three cigarettes.

Plasma fibrinopeptide A and beta-thromboglobulin in patients with chest pain.

Plasma levels of beta-thromboglobulin (BTG) and fibrinopeptide A (FPA), markers of platelet release and thrombin generation respectively, were measured in 48 patients within 3 days of admission to hospital for acute chest pain. Twenty-one patients had a confirmed myocardial infarction (MI); 15 had unstable angina without infarction; and 12 had chest pain due to noncardiac causes. FPA and BTG were also measured in 23 control hospital patients of similar age. Mean plasma BTG levels were not significantly different in the 4 groups. Mean plasma FPA levels were significantly higher in all 3 groups with acute chest pain when compared to the control subjects (p less than 0.01), but there were no significant differences between the 3 groups. Increased FPA levels in patients with acute chest pain are not specific for myocardial infarction, nor for ischaemic chest pain.

Factor IX thrombogenicity: in vivo effects on coagulation activation and a case report of disseminated intravascular coagulation.

An episode of defibrination with bleeding following high dose Edinburgh Factor IX (D.E.F.IX) replacement in a patient with haemophilia B undergoing knee joint replacement is reported. We have also monitored plasma fibrinopeptide A levels in patients with haemophilia B following ten standard doses of D.E.F.IX (15-20 u/kg) and have been unable to document any change. Activation of the coagulation system, as previously noted, appears to be related to the use of Factor IX concentrates in high doses.

Low dose heparin in the prevention of deep-vein thrombosis after aortic bifurcation graft surgery.

In a randomised double-blind controlled trial 24 patients undergoing elective aortic bifurcation graft surgery received subcutaneous calcium heparin (2,500 u pre-operatively then 5,000 u 12-hourly for 7 days) and 25 control patients received saline injections. All patients received a routine dose of intravenous sodium heparin intra-operatively. The trial was terminated because of excess bleeding complications in patients receiving subcutaneous heparin (8 vs. 1, p less than 0.05). Deep vein thrombosis occurred in 6 control patients and 2 patients on heparin (p less than 0.05). In this group of patients undergoing major vascular surgery the risk of bleeding due to heparin outweighed the potential benefit of thrombotic prophylaxis.

A comparative study of the effects of adrenoceptor antagonists on platelet aggregation and thromboxane generation.

Platelet aggregation and thromboxane A2 have been implicated in the pathogenesis of several forms of vascular disease. The aim of this study was to determine the effect of a wide range of adrenoceptor antagonists on platelet aggregation, and thromboxane A2 production, from normal human platelet rich plasma in vitro. Labetalol, pindolol and propranolol inhibited platelet aggregation to collagen in a dose dependent manner. Increasing the concentration of collagen "shifted" the dose response curve to the right. These 3 drugs also significantly inhibited thromboxane A2 generation in response to collagen but not to arachidonic acid. This effect was independent of any inhibitory effect of these drugs on platelet aggregation, and occurred at a drug concentration close to that obtained in vivo. Atenolol, metoprolol, prazosin and timolol were similarly assessed but had no effect on either platelet aggregation or thromboxane A2 generation. This ability of labetalol, pindolol, and propranolol to inhibit platelet aggregation and thromboxane generation, may be of clinical benefit in view of the increasing evidence implicating thromboxane A2 in the pathogenesis of vascular disease.

A comparison of spontaneous platelet aggregation in whole blood with platelet rich plasma: additional evidence for the role of ADP.

ADP, generated from red blood cells is believed to be responsible for the spontaneous aggregation of platelets in whole blood. This notion is based mainly on the use of enzymes which remove ADP. We have studied spontaneous platelet aggregation in whole blood and autologous platelet rich plasma obtained from 12 healthy male and female volunteers. Platelet aggregation was quantitated by measuring the fall in the number of single platelets counted using a whole blood platelet counter (Ultra Flo 100). In a rotating tube model, the mean fall in the number of platelets due to spontaneous aggregation was 56% in whole blood but, only 3% in platelet rich plasma prepared from the same blood samples. Spontaneous platelet aggregation in whole blood was unaffected by apyrase grade I, but was reduced to 15% by apyrase grade II, to 38% by creatine phosphokinase/creatine phosphate and to 9% by pyruvate kinase/phosphoenolpyruvate. The results of this study provide additional evidence that ADP generated in whole blood triggers the spontaneous aggregation of platelets.

White blood cell count and haematocrit as predictors of coronary recurrence after myocardial infarction.

Baseline white blood cell count (WCC) and haematocrit were examined in relation to recurrent coronary events and to all-cause mortality in 2026 persons enrolled in the first Persantin-Aspirin Reinfarction Study (PARIS-1) 2-60 months after myocardial infarction. WCC was strongly related to coronary recurrence (relative risk 3.5 for men with WCC greater than or equal to 9 X 10(9)/l vs men with WCC less than 5 X 10(9)/l) and total mortality (relative risk 2.6). No such relationships were found for haematocrit. WCC correlated also with cigarette-smoking, diuretic use, serum cholesterol and uric acid; however, the associations with coronary recurrence and total mortality persisted on multiple linear and logistic regression analysis including these variables and treatment group (p less than 0.001). WCC is therefore an easily-measured prognostic variable in survivors of myocardial infarction. Furthermore, we suggest that white blood cells may promote myocardial ischaemia by capillary plugging and/or release of toxic oxygen metabolites.

A critical investigation into the existence of circulating platelet aggregates.

By a method of counting single platelets in diluted whole blood, platelet aggregates were quantified ex-vivo. Four groups: 20 thrombotic patients, 10 non-thrombotic patients, 10 healthy old controls and 10 healthy young controls were included in the study. Using a 19 gauge needle, with and without tubing, venous blood was taken into buffered EDTA, as a disaggregating agent and buffered EDTA-formalin, as the fixative. The amount of platelet aggregates quantified was affected by the quality of venepuncture or the rate of blood flow through the needle, but was unaffected by the presence of the tubing. There was no statistically significant difference between the four groups, in terms of the platelet aggregates quantified, but scanning electron microscopy revealed the presence of irreversible aggregates, composed of platelet red and white blood cells, in the blood of a greater number of thrombotic patients than non-thrombotic or healthy controls. Platelet aggregates were also quantified in aliquots of platelet rich plasma, and were found to be significantly greater than the corresponding values in whole blood. The difference appeared to be due to increased viscosity of the plasma, induced by the fixative which reduces platelet mobility during centrifugation. It is concluded that the platelet aggregates which disaggregate in buffered EDTA may represent an artifact of blood collection; the irreversible aggregates are suspected to represent the in vivo circulating aggregates.