Enhanced spontaneous thrombolysis: a new therapeutic challenge.

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.

Coronary artery surgery is associated with different forms of atherogenic lipoprotein modifications.

BACKGROUND: 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. METHODS: 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. RESULTS: Preoperative plasma lipid peroxide concentration (2.2 micromol/L) increased after cardiopulmonary bypass (peak, 7.5 micromol/L; p<0.001) and remained significantly elevated at 3 months after surgery (4.2 micromol/L; p<0.01). 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. CONCLUSIONS: 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.

Inhibition of vascular smooth muscle cell migration by intact endothelium is nitric oxide-mediated: interference by oxidised low density lipoproteins.

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 N(G)-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.

Elevated plasma lipid hydroperoxides in patients with coronary artery disease.

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.

Evidence against hypercoagulability in coronary artery disease.

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 > or = 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.

Lipid peroxidation by activated platelets: a possible link between thrombosis and atherogenesis.

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.

Thrombotic status analyser. Measurement of platelet-rich thrombus formation and lysis in native blood.

A 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.

Different anticoagulants and platelet reactivity in cardiac surgical patients.

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.

Effect of leucocyte products on platelet thrombus formation, coagulation and spontaneous thrombolysis, as measured from native human blood, in vitro.

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.

Inhibition of platelet function by heparin. An etiologic factor in postbypass hemorrhage.

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.

Reduction of heparin binding to and inhibition of platelets by aprotinin.

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) (p < 0.0001). 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.

Effect of warfarin on arterial thrombogenesis: problems of monitoring.

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.

Enhanced platelet reactivity with erythropoietin but not following transfusion in dialysis patients.

Although the haemostatic defects that occur in uraemia are complex, a major factor is the anaemia of renal failure. This may now be corrected by recombinant human erythropoietin (rHuEpo) therapy rather than by repeated blood transfusion. Platelet reactivity to shear stress and collagen was measured using non-anticoagulated blood to study the effect of erythropoietin or blood transfusion on platelet function. Twenty dialysis patients were commenced on 25-50 U/kg rHuEpo twice weekly. The dose was adjusted after 3 months to maintain target Hb 10-10.5 g/dl. A further 15 patients were studied before and 10-12 days after receiving blood transfusion. Baseline platelet reactivity was subnormal in both groups versus control (P < 0.0001). In the rHuEpo group, a significant increase in platelet reactivity was observed at 2 months (P < 0.005) which disappeared at 3 months. This was not related to the increase in Hb (7.3 +/- 0.3 to 10.2 +/- 0.3 g/dl, P < 0.0001). There was no change in platelet reactivity after transfusion, despite an increase in Hb (6.2 +/- 0.2 to 8.8 +/- 0.2 g/dl, P < 0.0001) similar to that occurring in the rHuEpo group. We conclude that after rHuEpo therapy but not after transfusion a transient increase in platelet reactivity occurs which is dissociated from changes in platelet and red cell numbers.

Enhanced platelet reactivity and hypercoagulability in the steady state of sickle cell anaemia.

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.

Effect of heparin on in vitro platelet reactivity in cardiac surgical patients--a comparative assessment by whole blood platelet aggregometry and haemostatometry.

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.

Significance of plasma fibrinogen in coronary arterial disease: marker or causative risk factor for arterial thrombosis?

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 (approximately 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.

Hemostatic abnormalities in Sneddon's syndrome.

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 overall 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, spontaneous 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 improvement of the hemostatic profile.

High-dose aspirin inhibits shear-induced platelet reaction involving thrombin generation.

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.