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Substantial evidence suggests that spontaneous thrombolytic activity of blood is an effective mechanism for protection against tissue damage that is the consequence of lasting arterial occlusion. Despite the generally held belief that coronary artery disease and stroke are preventable, a promising avenue of prevention, namely the identification and prophylactic treatment of those at risk of inefficient spontaneous thrombolysis, has been neglected. This is mainly due to the lack of physiologically relevant test. A recently described technique allows the measurement of spontaneous thrombolysis, that is, lysis of an autologous platelet-rich thrombus in the absence of added plasminogen activators. Early results suggest that this test may have significant clinical potential both in identifying those at risk from fatal thrombotic event and in finding new therapeutic avenues of improving spontaneous thrombolytic activity.

This is the first laboratory evaluation of a new instrument, designed to test both platelet function and thrombolytic activity from a native blood sample, in vitro. The inventor assumed that the reduction and arrest of blood flow was due to activation, aggregation and stabilized thrombus formation by shear-activated platelets, and that re-establishment of flow was due to thrombolysis. Morphologic and functional studies presented here confirm these mechanisms. In vitro tests provided incontestable evidence for the principal role of platelets in the obstruction of flow (occlusion time) and for thrombolysis as the principal mechanism underlying the restoration of blood flow (lysis time). In addition to aggregation, it is the explosive generation of thrombin by shear-activated platelets that results in the formation of an occlusive haemostatic thrombus. Anticoagulation of blood completely prevented occlusion. Platelet-rich thrombus formation (occlusion time) was dose-dependently inhibited by monoclonal antibody against platelet glycoprotein (GP) Ib (6B4 and 12E4), aurin tricarboxylic acid, monoclonal antibody against platelet GPIIb/IIIa (MA-16N7C2 and abciximab), a synthetic GPIIb/IIIa antagonist (TAK-029), thrombin inhibitor (argatroban), and anti-von Willebrand factor, but not by anti-fibrinogen. Plasminogen activator streptokinase (Varidase) and tissue-type plasminogen activator (Monteplase) dose-dependently enhanced thrombolysis (lysis time) without affecting platelet function (occlusion time). The test is specific for thrombolysis. The plasmin inhibitor tranexamic acid prevented plasminogen activator-induced thrombolysis, while inhibition of clot retraction by cytochalasin B did not affect the lysis time. This rapid and sensitive global test of platelet function and thrombolytic activity could be of great value both in research and in clinical practice.

SummaryA physiologically relevant global in vitro test is described which allows the overall assessment of both thrombotic and thrombolytic activities of blood. In principle, native blood is drawn in pulses through a capillary tube where haemodynamic forces induce a platelet reaction culminating in vessel occlusion. Dislodgement/disintegration of the stabilised thrombus under pressure is a reflection of thrombolysis. Evidence is presented for the platelet-rich nature of the occlusion and that disruption of the thrombus and re-established patency is the result of thrombolysis, that is fibrinolysis with significant contribution from platelets.This test sensitively detects hypercoagulability (stasis); platelet hyperreactivity (coronary artery disease); anti-platelet effect (aspirin, prostacyclin) and the thrombolytic effect of, thrombin generation by, and resistance to streptokinase. Therefore, this overall assessment of thrombotic status could be of great diagnostic and therapeutic benefit in clinical practice.

Contribution of leucocytes to formation and lysis of arterial (platelet) thrombi was investigated. Secretory products of polymorphonuclear leucocytes (PMNs) during phagocytosis and cell lysates were prepared from eight volunteers. Platelet-rich thrombi were formed in flowing native human blood either by shear-stress or by collagen fibre, by haemostatometry. Tested in eight volunteers, PMN products significantly enhanced both thrombotic reactions and induced lysis of these thrombi. A specific inhibitor of leucocyte proteases (eglin c) inhibited platelet reaction to shear-stress and collagen and spontaneous thrombolysis. Our findings provide further evidence for the prothrombotic and potent thrombolytic effect of leucocytes associated with an arterial thrombus.

The effect of heparin (5 U/ml) on platelet function was examined by hemostatometry in vitro. A wide individual variation of this effect was found in 290 patients who underwent cardiac operations: 8.6% (25) experienced a proaggregatory effect, 58.6% (170) experienced a mild to moderate inhibition of platelet function, and 32.8% (95) experienced a severe inhibition. No significant difference was found among patient characteristics, including antiplatelet medication, in these three subgroups. In vitro measurements correlated significantly with ex vivo measurements, that is, from blood taken after heparinization (p < 0.0001; r = 0.97, n = 15). In 111 patients who underwent cardiac surgical intervention, a significant correlation (p < 0.0001; 0.4 < r < 0.52) was found between preoperative measurements of the degree of inhibition of platelet function by heparin and the total postoperative blood loss after 4, 12, and 18 hours. Similarly, there was a significant difference (p < 0.0001) in the total blood loss at 4, 12, and 18 hours between the subgroups that showed, in vitro, a mild to moderate inhibition of platelet function preoperatively compared with a severe inhibition (713 +/- 43 ml versus 1172 +/- 76 ml at 18 hours). It is concluded that platelet inhibition as a result of heparin varies among patients and appears to be a previously unrecognized etiologic factor in bleeding after cardiopulmonary bypass.

Platelet reactivity to shear stress and collagen and dynamic overall coagulation were measured in vitro from nonanticoagulated blood of 137 patients on warfarin. One hundred five matched, healthy subjects served as controls. Platelet reactivity to both stimuli and contribution of platelets to plasmatic coagulation were significantly inhibited in patients on warfarin. No correlation was found between platelet reactivity and the coagulation status assessed by the international normalized prothrombin time ratio (INR). Despite similar INR, platelet reactivity showed great individual variation. In 98 patients who were followed up for 3 months, measurement of platelet reactivity to shear stress could discriminate between those who had either bleeding or thromboembolic episodes. These findings suggest that monitored platelet function would help in individualizing oral anticoagulant regimens and hence would increase the benefit of therapy without the risk of bleeding complications. © 1993 Wiley-Liss, Inc.

A prospective controlled study was undertaken to investigate the haemostatic and coagulation status of 18 adult subjects in the steady state of sickle cell anaemia (SCA), using a relatively new in vitro technique. Shear induced haemostasis, whole blood dynamic coagulation, and spontaneous thrombolysis were measured using nonanticoagulated blood. As expected, the haemoglobin levels were significantly lower and platelet counts significantly higher in subjects with SCA compared with controls. Haemostasis and coagulation were significantly enhanced in SCA. No correlation was found between the raised platelet count and enhanced haemostasis or the reduced haemoglobin and hypercoagulation, respectively. Hyperactivity of the haemostatic system may have a pathogenic role in vaso-occlusive microthrombotic events and in the leg ulcers, both of which occur frequently in SCA.

The relationship between fibrinogen and severity of disease was measured in patients with coronary arterial disease ( n = 301) prior to surgical coronary revascularisation. Platelet reactivity (shear-induced haemostasis) was measured from non-anticoagulated blood, in vitro. Coagulation was assessed by the clotting time of flowing native blood (dynamic) and by the conventional (stagnant) tube tests. Significantly enhanced platelet reactivity to shear-stress was observed when patients with one-vessel disease were compared to those with two- or three-vessel disease ( P = 0.003). Neither coagulation nor fibrinogen were significantly related to the severity of disease. Furthermore, patients who had myocardial infarction ( n = 144) showed enhanced platelet reactivity ( P = 0.02) as compared to those who had not ( n = 157). Again, neither coagulation nor fibrinogen discriminated between these groups of patients. Relationship between plasma fibrinogen and platelet reactivity was also investigated in vitro. Identical blood samples with normal (220–280 mg/dl) and elevated plasma fibrinogen (≈ 500 mg/dl) were compared by measuring platelet reactivity and coagulation from native blood and platelet aggregation in whole blood. The in vitro studies suggested that plasma fibrinogen and platelet reactivity are inversely associated. Furthermore, increased fibrinogen prolonged dynamic coagulation. These findings do not support the assertion that elevated plasma fibrinogen is a true causative factor for coronary arterial disease and arterial thrombosis.

BACKGROUND A unifying concept of explaining all pharmacological actions of aspirin by the irreversible blockage of the enzyme cyclooxygenase and therefore the inhibition of prostaglandin biosynthesis has left many unanswered questions. METHODS AND RESULTS Two hundred ninety-four patients taking 75 mg/day aspirin were tested 3 months after coronary artery bypass surgery. Platelet thromboxane formation (whole blood aggregation to arachidonate) was completely prevented in 80% of patients. Compared with matched healthy controls (n = 95), a significant platelet hyperreactivity was observed in patients (p less than 0.0001 versus less than 0.002). Ninety patients were advised to increase their daily dose of aspirin from 75 mg to 300 mg. Platelet reactivity retested 1 month after increasing the dose has significantly decreased (p = 0.0008; less than 0.0001), whereas it remained unchanged in those patients (n = 84) who continued with the same dose regimens. In normal subjects, ingestion of a single 600-mg aspirin significantly inhibited shear-induced platelet reaction. CONCLUSIONS It is concluded that aspirin does affect the platelet response to shear forces, but this requires higher dosage (greater than 300 mg/day), suggesting a mechanism probably different from that of interference with thromboxane formation.

A broad-based, dynamic model of intrinsic coagulation is described. Non-anticoagulated whole blood was perfused through polyethylene tubing under standard conditions, and coagulation (cessation of flow) was monitored by pressure changes. The dynamic coagulation test (DCT) is a sequel to the shear-induced haemostasis, a platelet function test routinely performed prior to coagulation. DCT has two important advantages over stagnant overall clotting tests: i./ DCT reflects platelet coagulant activities; selective activation by adenosine diphosphate or shear-stress or inhibition of platelets by prostacyclin greatly enhanced or prolonged dynamic coagulation, respectively. Furthermore, activation of platelets by plasminogen activators (streptokinase, t-PA) was manifested in a significantly shortened coagulation. ii./ DCT allows the rapid assessment of fibrin crosslinking, the mechanical stability of the clot formed. Antibody against factor XIIIa greatly prolonged the time until completion of clotting. In patients taking oral anticoagulant (n=54), strong correlations were observed between DCT, the prothrombin time (INR) and the thrombelastograph measurements. It is concluded that this simple assay could be useful in the overall screening for coagulation abnormalities.

purpose: Prostacyclin, a potent inhibitor of platelet function and vasodilator, has been used to treat peripheral vascular disease. The aim of this study was to monitor the thrombotic status of patients treated by infusion of a stable prostacyclin analogue, iloprost. patients and methods: Thirteen patients with peripheral vascular disease underwent iloprost infusion for 3 days (8 hours each day) in a dose ranging from 0.5 to 2 ng/kg/minute. Variable parameters of thrombosis such as platelet reactivity (shear-induced hemostatic plug formation and thrombus formation on a collagen fiber), coagulation, and spontaneous thrombolysis (dislodgment of hemostatic plugs) were measured from non-anticoagulated blood samples by hemostatometry immediately before and 1 hour after the infusion and on the last day, 4 hours after initiation of the infusion. results: Analysis of data from all patients 1 hour after the infusion showed no changes in platelet reactivity and spontaneous thrombolysis, but coagulation was significantly enhanced. In four patients, significant platelet hyperreactivity was observed after the infusion. Four of the five patients tested while undergoing iloprost infusion showed an enhanced thrombotic reaction and markedly enhanced coagulation. Iloprost employed in vitro in a concentration that corresponds to the therapeutic peak blood level caused no inhibition of platelet function but significantly enhanced coagulation. The threshold in vitro iloprost concentration at which antiplatelet effect and increased spontaneous thrombolysis were observed was twice that of the therapeutic blood level. conclusions: These findings challenge the view that antagonism of platelet function is an important factor of iloprost therapy. Furthermore, platelet hyperreactivity in some patients and markedly enhanced coagulation during and after infusion of iloprost in general, represent a risk of thromboembolism, especially as patients are already in a prethrombotic condition.

Spontaneous thrombolysis is an endogenous protective mechanism against lasting arterial thrombotic occlusion, which is implicated in the pathogenesis of myocardial infarction and acute coronary events. Novel therapies for coronary heart disease (CHD) targeting atherosclerosis and thrombosis, together with cardiovascular prevention programs targeting risk-factors and lifestyle provide evidence that CHD is preventable. Although reduced fibrinolytic activity is a recognized risk-factor for ischemic cardiovascular events, it has so far been neglected. Our knowledge of the fibrinolytic effect of drugs commonly used for CHD such as antiplatelet agents (aspirin, ticlopidine, clopidogrel), anti-diabetic biguanides (phenformin, metformin) or anti-hypertensive drugs is scanty and conflicting. This is mainly due to the lack of a global test of spontaneous thrombolysis, as opposed to fibrinolysis of plasma or whole blood, i.e. the assessment of various activators and inhibitors of the fibrinolytic system. A recently described technique allows the measurement of spontaneous thrombolysis, that is, lysis of an autologous platelet-rich thrombus in the absence of added plasminogen activators. Early results suggest that this test may have significant clinical potential both in identifying those at risk of fatal cardiac events and in finding new therapeutic avenues or lifestyles to improve spontaneous thrombolytic activity.

The historical development of in-vitro bleeding time tests, using solely shear forces to initiate haemostatic plug formation, in the absence of the vessel wall or its components, is described. Techniques that have no potential for routine use in clinical practice, such as flow chambers and cone-and-plate viscometers, are excluded. The problems related to the use of citrated blood in platelet function tests are discussed. In light of the pivotal role of thrombin and platelet-dependent thrombin generation in haemostasis/thrombosis, the advantage and clinical benefits of testing unadulterated native blood is discussed.

Increasing evidence shows that thrombogenicity and atherogenicity of lipoproteins are related to modifications involving oxidative, enzymatic, or physical alterations of these molecules. Findings on lipid peroxidation associated with cardiopulmonary bypass are conflicting, and the possible other forms of atherogenic lipid modification are unknown. The various forms of lipoprotein modifications including lipid peroxidation, desialylation, and leukocytic elastase activity after coronary artery bypass graft operations are examined.In patients undergoing coronary artery bypass graft operations, plasma total lipid hydroperoxides (n = 102), plasma leukocytic elastase activity (n = 125), free radical formation (n = 30), low-density lipoprotein oxidation, and sialic acid content before operation and at 2, 24, 48, and 72 hours after cardiopulmonary bypass and 3 months after operation were measured.Preoperative plasma lipid peroxide concentration (2.2 micromol/L) increased after cardiopulmonary bypass (peak, 7.5 micromol/L; p0.001) and remained significantly elevated at 3 months after surgery (4.2 micromol/L; p0.01). There was a significant correlation between increased free radical generation and lipid peroxide levels in blood at all postoperative intervals. Low-density lipoprotein separated from plasma samples showed increased oxidation 48 hours after bypass. Sialic acid content of low-density lipoprotein was significantly reduced 48 hours after bypass. Plasma elastase activity increased significantly at all postoperative intervals.Coronary artery bypass graft operation is associated with generation of sustained blood levels of modified lipoproteins. These thrombogenic and atherogenic particles may play an important role in hemostatic and arteriosclerotic complications of coronary artery bypass graft operations.

Migration of vascular smooth muscle cells (SMCs) leading to neointimal hyperplasia is an early and cardinal feature of atherogenesis. Migration of rat aortic SMCs from an upper chamber towards a lower one has been studied in a microchemotaxis (Boyden) chamber. Spontaneous migration of SMCs was practically prevented by the presence of endothelium in the lower chamber and was reduced if endothelial cells were substituted with endothelial cell-conditioned medium. Endothelial cells which had been treated with either the inhibitor of protein synthesis cycloheximide or nitric oxide synthesis NG-nitro-L-arginine showed no inhibitory effect on SMC migration. Addition of a nitric oxide donor S-nitroso-N-acetylpenicillamine to cell-free medium in the lower chamber prevented SMC migration. Addition of native LDL to endothelial cells had no effect on SMC migration, while (UV light) oxidised LDL completely abolished the inhibitory effect of endothelial cells on SMC migration. It is concluded that via nitric oxide, endothelium exerts a powerful inhibitory effect on SMC migration. This effect of intact endothelium is completely abolished by oxidised LDL applied in a concentration, which is relevant to those measured in plasma of patients with severe coronary artery disease. It is suggested that oxidised LDL may contribute to the pathogenesis of atherogenesis by stimulating migration of SMCs from media to the intima via abolishing the physiological inhibitory effect of normal endothelium.

Sustained presence of lipid peroxides in the circulation and their plasma carrier is a controversial issue. Particularly, there is no firm evidence for an increased plasma lipid peroxide level in patients with atherosclerosis. In this study, a strong correlation was found between plasma total lipid hydroperoxide and lipid hydroperoxide content of LDL cholesterol ( r = 0.882; p < 0.001; n = 16). Lipid hydroperoxides in plasma were carried almost exclusively (89%) in LDL. In 70 patients tested 3 months after coronary artery bypass graft surgery with a specific assay, plasma lipid hydroperoxide levels were significantly increased when compared with matched healthy controls (4.31 ± 0.23 nmol/ml and 2.34 ± 0.13 nmol/ml, p < 0.0001, patients vs controls, respectively). These concentrations are 10 times lower than those detected by the nonspecific thiobarbituric acid assay. However, considering the in vitro concentration range in which oxidized LDL exerts important atherogenic effects, the elevated plasma lipid hydroperoxide levels measured in atherosclerotic patients have pathologic significance. (Am Heart J 1997;134:572-6.)

The strong epidemiological association between elevated plasma clotting factors and coronary artery disease is generally interpreted as evidence that patients with coronary atherosclerosis are in a procoagulant (hypercoagulable) state. A dynamic global test was used to assess the overall coagulation status of 761 patients with coronary artery disease scheduled for coronary artery bypass grafting and compared to healthy matched controls (n = 100). Platelet reactivity to shear-stress was simultaneously measured from identical, non-anticoagulated blood samples. Contrary to expectation, the overall coagulation in cardiac patients did not differ significantly from that of controls. Furthermore, the coagulation status of patients bore no relationship to the severity of coronary atherosclerosis. The latter is in contrast with platelet reactivities, which were significantly increased in patients with ≥2 vessel disease as compared with single vessel disease. The present results do not necessarily conflict with the finding of elevated plasma clotting factors in cardiac patients. However, they do not support the claim that these markers are a reflection of a hypercoagulable state. Indeed, this study confirms that such patients are in a prothrombotic state, which is related to enhanced platelet reactivities, and not to a prothrombotic imbalance of the coagulation mechanism.

Generation of reactive oxygen species (ROS) by platelets was detected by a cyano-tetrazolium dye (CTC) which forms fluorescent formazan on the cell surface upon reduction. Fluorescence was quantitated by a densitometric device. Resting platelets in plasma produced significant fluorescence (P < 0.0001), and addition of thrombin enhanced the fluorescence of the coagulated platelet mass even further (by 2 h, a 6- and 8-fold increase over fluorescence of platelet-free plasma was measured, respectively). Blood containing CTC was perfused through a glass capillary tubing and the action of shear forces resulted in the formation of an occlusive platelet thrombus. Such thrombi (formed either from whole blood or platelet-rich plasma) were intensely fluorescent, indicating formation of ROS in the platelet mass (a 10- and 8-fold increase in fluorescence over coagulated plasma, respectively). Lipid peroxide content of resting platelets in platelet-rich plasma was doubled over 24 h storage, while addition of thrombin caused a 7.4-fold increase (P < 0.0001) of lipid peroxides in the retracted platelet-rich plasma-clot. Transition metal chelator and antioxidant prevented lipid peroxidation by platelets in response to thrombin. Thrombin activation of (washed) platelets in plasma-free medium caused only 1.4-fold increase in oxidation of added low density lipoprotein (LDL). In contrast, thrombin activation of platelets suspended in de-lipidated autologous plasma resulted in a 5.25-fold increase (P < 0.0001) in LDL oxidation. Generation of ROS and lipid peroxides by platelets can be an important mechanism through which thrombotic events contribute to atherogenesis.

A technique for assessing platelet reactivity to shear stress from nonanticoagulated blood samples was employed to compare the relative effects of an unfractionated heparin, a low-molecular-weight heparin, and hirudin. The in vitro platelet effect of unfractionated heparin (5 U/mL) was measured in 290, the effect of a low-molecular-weight heparin (1 anti-Xa unit/mL) in 74, and the effect of hirudin (8 micrograms/mL) in 50 cardiac surgical patients. The relative proportions of patients exhibiting an enhanced platelet reactivity, a mild to moderate inhibition, and a severe inhibition were, respectively: 8.6%, 58.6%, and 32.8% for unfractionated heparin; 22%, 66%, and 12% for the low-molecular-weight heparin; and 6%, 66%, and 28% for hirudin. At the concentrations examined, a significantly greater proportion (p < 0.01) of the patients exhibited enhanced platelet reactivity and a significantly smaller proportion (p < 0.01) showed severely inhibited platelet reactivity associated with the low-molecular-weight heparin versus the unfractionated heparin, whereas there was no significant difference between the patients treated with hirudin and unfractionated heparin. Although the relevance of this study is limited because the clinically appropriate concentration of the alternative anticoagulants and comparative doses are unknown, it can be inferred that low-molecular-weight heparin may reduce the blood loss associated with cardiopulmonary bypass.

The direct effect of aprotinin on in vitro platelet function was assessed by hemostatometry (n = 10). No significant enhancement was demonstrated. However, aprotinin reduced platelet inhibition secondary to heparin. Hemostatometry demonstrated a significant preservation of in vitro platelet function (n = 25) (p = 0.04), which was particularly marked (p = 0.003) in the subgroup (n = 7) demonstrating a severe inhibition of platelet function with heparin. Aprotinin significantly reduced the binding of tritium-labeled heparin to both nonactivated (n = 25) (p = 0.004) and activated platelets (n = 25) (p0.0001). We conclude that interference with heparin-induced inhibition of platelet function by aprotinin may be one of its hemostatic actions in cardiac surgery. This effect is probably secondary to aprotinin reducing binding of heparin to platelets.

The in vitro effect of heparin on platelet reactivity was assessed simultaneously by haemostatometry (response to shear stress) and whole blood platelet aggregometry response to collagen (WBPA). From each blood sample a ratio (HR for haemostatometry and MR and IR for WBPA) showing platelet reactivity in the presence or absence of heparin (5 U/ml) was calculated. A value less than 1 represented a proaggregatory effect and greater than 1 an inhibitory effect. Non-anticoagulated blood samples obtained from 290 cardiac surgical patients were tested by haemostatometry and citrated whole blood samples from 100 patients with aggregometry. Haemostatometry demonstrated a proaggregatory effect of heparin in 8.6% (25) and an inhibitory effect in 91.4% (265). Assessed by WBPA, heparin was proaggregatory in 41-46% and inhibitory in 54-59%. In the 100 patients tested by both methods there was a significant correlation between the findings with the two techniques (r = 0.46, p less than 0.0001). A wide individual variation in the platelet effect of heparin was demonstrated. This variation appeared greater and a higher proportion showed inhibition when blood was tested by haemostatometry.

Sneddon's syndrome is a rare condition comprising widespread livedo retucularis and multiple episodes of transient cerebral ischemia. Treatment to date has been empirical. The hemostatic/thrombotic status of 4 patients with Sneddon's syndrome was studied by a unique technique, hemostatometry, which measures primary hemostasis (shear-induced platelet plug formation), the over all coagulation, and thrombolysis (dislodgment of the hemostatic plugs) from nonanticoagulated blood. In all 4 patients, platelet reactivity, which shows itself in the initial phase of the hemostatic reaction, was enhanced. The overall hemostasis, in which the generation of thrombin by activated platelets plays the decisive role, was enhanced in 3 patients. Three of the 4 patients had hypercoagulation, and in 3, spontane ous thrombolysis was inhibited. Treatment was commenced with aspirin and nifedipine, and patients were monitored both clinically and by serial hemostatometry over two years. One patient had one further transient ischemic episode; the other 3 remained asymptomatic. Thus, the observed clinical improvement correlated with im provement of the hemostatic profile.

Bleeding due to impaired primary haemostasis is common in uraemia. However, thrombo-embolic episodes are also a clinical problem in dialysis patients. Platelet reactivity to shear stress (haemostasis, H1 and H2), exposure to collagen fibre (thrombus growth) and coagulation of flowing blood (clotting time, CT1 and CT2) were measured in non-anticoagulated blood samples taken immediately before and 18-24 h after haemodialysis (n = 26) and from patients maintained on continuous ambulatory peritoneal dialysis (CAPD, n = 30). H1 (p < 0.001), H2 (p < 0.01), percent thrombus growth rate (p < 0.03), CT1 (p < 0.01 and CT2 (p < 0.05) were restored towards normal after haemodialysis. Results obtained in the CAPD patients demonstrated that the mean values for formation of the haemostatic plug lay between the pre- and posthaemodialysis values; however, CT1 (p < 0.01) and CT2 (p < 0.05) were prolonged in CAPD compared with values after haemodialysis. These data, which indicate platelet function from non-anticoagulated blood and coagulation under flow conditions, (1) confirm that there is impaired haemostasis in uraemia; (2) demonstrate an improvement in haemostasis after haemodialysis; (3) show that peritoneal dialysis results in a haemostatic profile which falls between the pre- and posthaemodialysis pattern, and (4) show that neither dialysis modality returns haemostasis to normal.

The rationale for predicting the risk of excessive postoperative bleeding by assessing the hemostatic status of a patient before cardiopulmonary bypass was investigated. A novel, rapid, overall test (hemostatometry) consisting of a physiologically relevant test of platelet function (shear-induced hemostasis) and coagulation was performed using nonanticoagulated blood and compared with the routine coagulation screen. Two hundred five patients undergoing elective coronary revascularization were studied 3 to 4 days before operation. Forty-nine bled excessively for nonsurgical reasons; none were predicted by the routine coagulation tests. Using a stepwise discriminant analysis, hemostatometry correctly predicted 31 of 49 (63%). Thirty of 156 predicted as bleeders by hemostatometry did not bleed. Thus, preoperative hemostatometry predicted 77% of the true outcome. The false predictions suggest, however, that certain bleeding abnormalities probably acquired during cardiopulmonary bypass cannot be predicted. These findings do not justify the routine use of preoperative tests in assessing the bleeding risk in patients undergoing cardiopulmonary bypass.

The sequelae of thrombus formation, both by shear forces and by collagen fiber, the subsequent coagulation, and the dislodgement of thrombi (thromboly sis) were measured from a small volume of nonanticoagulated blood sample, by a new instrument. Addition of streptokinase (SK) or tissue-type plasminogen activator (rt-PA) to the blood sample eliminated the need for anticoagulation for thrombolysis measurement: clot lysis preceded and allowed thrombolysis to occur. The test revealed activation of platelets and coagulation by SK and rt-PA. Apart from this general trend, platelet reactivity in response to plasminogen activator showed great individual variation. Whether the greatly enhanced (42 %) or prolonged hemostasis (13%), observed in different blood samples with rt-PA, could be used as a predictor of reocclusion or bleeding complications remains to be established. Thrombolysis did not occur in 12 % of the samples tested. These thrombolysis models may be useful for developing new agents for the dissolution of platelet-rich thrombi. It is suggested that this technique be used for monitoring thrombolytic therapy.

Two in vitro models of coronary thrombolysis in man, i.e. dislodgement of thrombi formed from non-anticoagulated human blood, either by (i) shear-stress or (ii) interaction of platelets with type I collagen fibre, were studied. Heparinization (1 U/ml) of blood prior to thrombus formation by (i) strongly inhibited spontaneous dislodgement (P less than 0.0001). Heparin (1 U/ml), when added with streptokinase (SK) or tissue-type plasminogen activator (rt-PA) prior to thrombus formation, considerably delayed thrombolysis. Furthermore, thrombolysis occurred much earlier when thrombi were perfused with SK or rt-PA in native than in heparinized blood. Heparin inhibited binding of 125I-rt-PA (17%, P less than 0.02) and plasminogen (88%, P less than 0.0005) to platelets activated by ADP in citrated platelet-rich plasma. We conclude that heparin interferes with the fibrinolytic system at the surface of activated platelets. Our findings suggest that heparin administration prior to thrombolytic therapy for acute myocardial infarction should be questioned.

Deformability and fragility of red blood cells (RBC) of established arthritic rats were measured by a filtration technique. They showed a significantly decreased deformability and, accordingly, a considerably increased fragility to micro heat stimulation, induced by laser irradiation. Subacute treatment of arthritic rats with aspirin and indomethacin had no effect on RBC behaviour whereas hydrocortisone restored the deformability of RBC almost to normal. Treatment of RBC of arthritic rats with neuraminidase caused the deformability of the cells to be restored to a supranormal level.

SummaryAddition of N-acetyl neuraminic acid (sialic acid, NANA) to citrated rat platelet-rich plasma significantly inhibited aggregation induced by near-threshold concentration of ADP, collagen or thrombin. In heparinized rat platelet-rich plasma aggregation of platelets induced by endotoxin or tumour cells of various origins was also inhibited by sialic acid.It is suggested that exogenous or endogenous sialic acid may act against various aggregating stimuli on the platelet membrane by masking a common factor through which various aggregating agents exert their effect.

1. Filterability (deformability) of erythrocytes of patients with Raynaud's phenomenon together with progressive systemic sclerosis (PSS) was decreased compared with the filtration of erythrocytes from normal subjects. 2. PSS erythrocytes showed lower electrophoretic velocity and about 23% less neuraminidase-removable sialic acid density on their surface than the normal erythrocytes. 3. PSS erythrocytes showed more adherence to cultured endothelial cells than the control normal erythrocytes. 4. It is concluded that the increased rigidity and adherence of PSS erythrocytes-5-have pathological significance in the mechanism of vascular abnormalities in PSS.

Ten patients with moderate to severe Raynaud's syndrome were recruited into a four week randomised double blind crossover study to compare the efficacy of UK-38,485 50 mg, a new thromboxane synthetase inhibitor with that of placebo. With the doses used there was no significant difference between the two treatment periods in the number, severity and duration of ischaemic attacks, the mean hand temperatures, forearm and digital blood flow and red blood cell rheology.

Thrombi formation resulting from laser-induced injury to mesenteric circulation has been studied in spontaneously hypertensive (SH) and normotensive rats. The thrombotic reaction was enhanced in both venules and arterioles of the SH rats. In the arterioles of the hypertensive rats, the vasoconstriction which develops downstream from the thrombus is greatly intensified and the time to form micro-vascular haemostatic plugs significantly decreased in the arterioles of these animals. These phenomena have been explained on the basis of an increased reactivity of the vascular smooth muscle of the SH rats to vasoconstrictor substances liberated from platelets at the site of injury.

We conducted a retrospective study to correlate thrombotic variables with the risk from an acute coronary event, using a new in vitro technique, which measures haemostasis, thrombolysis and coagulation from non-anticoagulated blood samples. The analysis was based on data from 63 patients who had undergone exercise radionuclide ventriculography, 50 of whom were considered to be at risk from an acute coronary event because they satisfied at least one of the following three criteria: (1) coronary angiography documented disease, (2) prior myocardial infarction, (3) ventriculography assessed provocable ischaemia. Fifty matched normal subjects were used as controls. Significantly enhanced haemostasis was measured in patients considered at risk from acute coronary event, and haemostatic activity was further increased in patients with provocable ischaemia. Haemostasis in eight patients at risk (provocable ischaemia), who continued with the medication during the test, did not differ significantly from the controls. Greatly reduced spontaneous thrombolytic activity was measured in all patients at risk from acute coronary events. These findings suggest the presence of hyperactive platelets in patients at risk from acute coronary events, with an additional risk of greatly reduced spontaneous thrombolytic activity.

The effects of prostacyclin (PGI2) on haemostasis and thrombolysis in human blood were investigated in vitro using the ‘Haemostatometer’. In freshly drawn native blood samples, 5 ng/ml PGI2 greatly inhibited haemostasis but did not influence clotting time. The effect was potentiated in heparinised blood: 1 ng/ml PGI2 effect on haemostasis was still detectable, and the duration of the effect was prolonged. Thrombolytic activity was tested in heparinised blood by first allowing haemostasis and then measuring the time until rebleeding occurred through the consolidated haemostatic plugs. PGI2 at 0.5 ng/ml had no significant effect on haemostasis, but significantly decreased the time for spontaneous thrombolysis (p < 0.001). Comparable effects were demonstrated in blood samples containing 50 U/ml strepto-kinase. Increased thrombolysis also occurred when haemostatic plugs formed from control blood were perfused with blood containing 5 ng/ml PGI2. The results suggest that PGI2 has a thrombolytic effect independent of any contribution from the vessel wall and that it is a more potent stimulator of thrombolysis than inhibitor of haemostasis.

The fine details of the in vivo technique for producing thrombus formation in microcirculation by HeNe laser are described. The criteria of assessing the thrombotic reaction in the mesenteric arteries and veins of mice and venules of the mesoappendix of rats are established. Validity of the test is demonstrated by two potent antiplatelet drugs, aspirin and Fludilat. The method, performed especially in mice, is highly suitable for screening compounds for antithrombotic effects.

Primary hemostasis (PH), i.e ., hemostatic platelet plug formation, and the subsequent coagulation were recorded and quantified from the same nonanticoagulated venous blood sample with the use of the Haemostatometer®. In addition, platelet thrombus formation induced by interaction of flowing native blood with a collagen fiber under low shear rates (450 s−1) was simultaneously analyzed by this device. The effect of monoclonal antibodies (MoAbs) directed against von Willebrand’s factor antigen (vWF: Ag), platelet glycoprotein Ib (GPIb) and the GPIIb/IIIa complex, andfibrinogenwere studied. PH was significantly inhibited by MoAbs against vWF:Ag, GPIIb/IIIa, and fibrinogen but was unaffected by antibody against GPIb. Collagen-induced thrombosis was prevented by MoAbs against vWF:Ag and GPIb, slightly inhibited by antifibrinogen, and unaffected by blockage of platelet membrane GPIIb/IIIa. The effect of a single 600-mg dose of aspirin was monitored, and abnormal PH was still detectable five days later. From the 13 hemophiliacs tested, 7 showed significantly prolonged PH. In von Willebrand’s disease, a characteristic defect of PH with significant inhibition or absence of collagen-platelet interaction was observed in all the 11 patients. PH was greatly prolonged in both of the two patients with storage pool deficiency. The technique detected improvement of platelet function, i.e ., PH in all of six patients with bleeding disorders after replacement therapy or DDAVP infusion. The authors conclude that the Haemostatometer technique is a sensitive test for determining platelet dysfunction and monitoring efficacy of factor-replacement or DDAVP therapy.

The 'filterability' and electrophoretic mobility of erythrocytes from 42 patients with systemic sclerosis and Raynaud's phenomenon were studied and compared with the findings from 24 patients with Raynaud's disease and 26 normal controls. Red blood cells from patients with systemic sclerosis and Raynaud's phenomenon were less filterable (P less than 0.0001) and had decreased electrophoretic mobility (P less than 0.001) compared with erythrocytes from patients with Raynaud's disease and the controls. There was no significant difference between the values from the patients with Raynaud's disease and the controls. These results indicate that measurement of erythrocyte filterability and electrophoretic mobility may be useful in the differentiation of patients with Raynaud's disease who have no underlying collagen disease from those who have Raynaud's phenomenon in association with systemic sclerosis.

Bronchial resistance was measured by the lung overflow technique in guinea pigs. Intravenous infusion of thrombin produced a significant increase of bronchial resistance, induced by platelet constituents released from the thrombi and deposited in the arteries and capillaries of the lung. This was evidenced by increase of bronchial resistance and mortality having been aggravated by e-aminocaproic acid (inhibition of fibrinolysis), by infusion of gelatin (sludge phenomenon) as well as by catecholamines (stress effect) and significantly inhibited by massive doses of heparin and by experimental thrombocytopenia. Emphasis is laid on the significance of pulmonary microembolism in the development of severe pulmonary hypertension and edema, the cause of which is difficult to diagnose clinically, yet demonstrable at autopsy. The recommended method is held to be suitable for the objective study of the process in animal experiment.