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Laboratory diagnosis of immune heparin-induced thrombocytopenia.

Immune heparin-induced thrombocytopenia (HIT) is a distinct immunohematologic syndrome in which laboratory detection of the pathogenic HIT antibodies is diagnostically useful. Assays can be broadly classified as platelet activation assays (which detect HIT antibodies based on their characteristic platelet-activating properties) and antigen assays (which measure antibodies reactive against platelet factor 4 complexed with heparin or other polyanions). Available tests vary considerably in their sensitivity-specificity profiles for detecting the antibodies and in their predictive values. The high sensitivity of certain assays means that HIT can be readily ruled out (high negative predictive value). However, because heparin-treated patients often generate nonpathogenic antibodies, a positive test does not necessarily indicate clinical HIT. Laboratory methods for detecting HIT antibodies have undergone an evolution similar to that of red blood cell serology and which parallels the Goldilocks tale, that is, progression from too insensitive (too hard) to too sensitive (too soft) to "just right." However, optimal diagnostic information requires that laboratory test results be interpreted in the appropriate clinical context.

Clinical Laboratory Techniques↗

Defective platelet beta-N-acetyl hexosaminidase content and release in chronic myeloproliferative disorders.

BACKGROUND AND OBJECTIVES: Abnormalities of platelet function or structure are a hallmark of chronic myeloproliferative disorders (MPD). In vivo platelet activation with the release of alpha- and delta-granules in the circulation is one of the most frequently described alterations in MPD. Platelets contain and release upon activation also lysosomes, and in particular beta-N-acetylhexosaminidase (Hex). We have assessed whether the content and in vivo release of Hex of platelets from MPD patients is altered. DESIGN AND METHODS: Twenty-three MPD patients were compared with 19 age- and sex-matched healthy controls. The activity of platelet beta-N-acetylhexosaminidase was measured in plasma, serum and in the capillary blood emerging from the skin wound inflicted for the measurement of the bleeding time. Lysosome integral membrane protein (LIMP or CD63), lysosome-associated membrane protein (LAMP-2 or CD107b) and P-selectin were evaluated by flow cytometry. Platelet aggregation in vitro and the release of beta-N-acetylhexosaminidase, ATP and beta-thromboglobulin were performed to study platelet reactivity. RESULTS: Hex levels in plasma were significantly higher in MPD than in controls while the release of Hex in the bleeding time blood, i.e. at a localized site of in vivo platelet plug formation, was lower in MPD and the platelet content of Hex was reduced. These changes were accompanied by in vivo platelet activation. Finally, the isoenzymatic pattern of Hex was altered in platelets of MPD patients, with a reduced amount of the Hex A isoform as compared with controls.b INTERPRETATIONS AND CONCLUSIONS: MPD patients present an altered platelet Hex content and release; prospective studies to assess whether altered platelet Hex is related to thrombotic/hemorrhagic complications and/or tissue fibrosis in MPD are warranted.

Adolescent↗

Platelet retention in coronary artery bypass surgery with and without a heart-lung machine. Cause of thrombosis in coronary artery bypass surgery.

The aim of this study was to examine platelet function after coronary artery bypass grafting (CABG) with and without the use of extracorporeal circulation (ECC). Sixteen male patients scheduled for CABG with (n = 8) and without (n = 8) ECC were included in the study. Platelet retention, as measured with a glass-bead retention test, was examined daily during the first postoperative week. Von Willebrand factor (vWF), ristocetin co-factor (Rcof) and prothrombin fragment (PF 1 + 2) were analyzed the day after the operation. We found a significant increase (p < 0.0001) in platelet retention during the first postoperative week after CABG. There was a tendency (not statistically significant) towards a more pronounced increase in the group operated on without ECC. This increase occurred despite the fact that all patients were treated with aspirin (75 mg daily) from the first postoperative day. The median time to maximal postoperative platelet retention was 2 days. In 3 patients platelet retention increased to more than 6 times the basal level.

Adult↗

Heparin-induced thrombocytopenia and thrombosis: a potential fatal complication in a routine treatment.

Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy. Life-threatening thromboembolism (HITT) may occur in a large number of patients with HIT. In this article diagnostic problems and the clinical course of 4 typical patients are described. Diagnosis was based on the occurrence of thrombocytopenia during heparin therapy and was confirmed in vitro by an ELISA to heparin-platelet factor 4 antibodies, heparin-induced platelet activation assay (HIPAA) or the platelet aggregation assay (PAA). Thrombotic complications developed in 2 patients, one of whom suffered a fatal embolism after accidentally rechallenging with low-dose heparin which was used to maintain the patency of an intravascular catheter. After discontinuation of heparin the thrombocyte count rapidly increased to normal values during treatment with the heparinoid danaparoid (Orgaran) without complications.

Aged↗

Thrombostatin inhibits cyclic flow variations in stenosed canine coronary arteries.

Thrombostatins are a group of compounds based upon a breakdown product of bradykinin, RPPGF. They inhibit alpha-thrombin-induced platelet activation by binding to protease activated receptor 1 and, at a lower affinity, by interacting with thrombin's active site. After a single intravenous infusion of MAP4-RPPGF (11.58 mg/kg), its t1/2alpha was 4.5 min with a clearance of 2.0 ml/min. MAP4-RPPGF administration had a sustained antiplatelet effect, preventing gamma-thrombin-induced (12.5 nM) platelet activation for 4 h. Its antiplatelet effect summated with that of aspirin and/or clopidogrel. MAP4-RPPGF was compared with aspirin and clopidogrel in the Folts model of coronary artery thrombosis. Dogs were randomized to 3 treatment groups: aspirin 1.14 mg/kg i.v., clopidogrel 0.5 mg/kg i.v., or MAP4-RPPGF 0.77 mg/kg i.v. Cyclic flow variations (CFV) were recorded in 5 untreated dogs hourly for 3 successive hours and for 1 h before (all groups >11 CFV/h), and for 2 h after drug infusion in each of the 3 treatment groups. After 1 h drug treatment, all groups of animals had <6 CFV/h; after 2 h treatment, all had <1 CFV/h. All agents significantly reduced CFV from control at each hour, but none was significantly better than any other. Thrombostatin was as effective as aspirin or clopidogrel in inhibiting coronary artery thrombosis in this canine model.

Animals↗

Evaluation of the cuticle bleeding time in canine haemophilia A.

In this paper we describe our clinical experience and results with the cuticle bleeding time test in a colony of cross-bred Labrador retrievers with severe haemophilia A. The dogs have a severe bleeding tendency with a high incidence of fatal haemorrhages in the central nervous system. Homozygous females appeared to be especially prone to this lethal complication. Factor VIII recovery and half-life determinations yielded results similar to the data from human studies. The cuticle bleeding time proved to be a good measure of the coagulation defect. The prolongation of the bleeding time could be completely abolished by administration of 10 to 15 units of canine factor VIII per kg body weight. We conclude that the cuticle bleeding time in canine haemophilia provides us with a suitable model for the in vivo study of new therapeutic materials.

Animals↗

Effect of timolol on platelet aggregation in coronary heart disease.

The immediate effect of beta-blockers versus placebo in platelet function was studied in ten healthy men one hour after either 40 mg propranolol, 5 mg timolol or placebo was given. Both beta-blockers had similar platelet effects. They increased the ADP-threshold in a platelet aggregation test and reduced the plasma level of beta-thromboglobulin whereas the primary aggregation remained unchanged. Forty-four patients were given timolol 10 mg twice daily or corresponding placebo medication in a secondary prevention trial after a myocardial infarction. At rest and also immediately after a near maximal exercise test the platelet function was similar on timolol and placebo. During exercise a lower ADP-threshold and a higher plasma level of beta-thromboglobulin was observed irrespective of whether the patients received placebo or timolol. Thus, the acute platelet inhibiting effect of timolol observed after a single dose disappeared during chronic timolol treatment. During 24 hours continuous ambulatory ECG monitoring patients on timolol treatment had significantly less arrhythmias than patients on placebo. A general effect on platelets cannot explain this antiarrhythmic effect of timolol during chronic therapy.

Adult↗

Heparin therapy leads to platelet activation and prolongation of PFA-100 closure time.

BACKGROUND: Heparin is used in the treatment of venous and arterial thromboembolic events, including unstable angina. Once unfractionated heparin is discontinued during the acute phase of unstable angina, it has been demonstrated that the disease process may be reactivated within hours. It is hypothesized that this reactivation may be a result of direct platelet activation by heparin that can linger even after the drug itself has been stopped. Prior studies have shown that heparin can either increase or decrease platelet activation. More recent studies have also shown conflicting effects of unfractionated heparin on PFA-100 testing. METHODS: We studied the in-vitro effects of unfractionated heparin on platelet function and PFA-100 testing. Unfractionated heparin was incubated with whole blood taken from 18 healthy volunteers. Platelet activation and aggregation was assessed with and without the presence of heparin. RESULTS: Platelet aggregation and activation were increased in the presence of heparin. Unfractionated heparin also significantly prolonged collagen/adenosine diphosphate closure time but did not affect collagen/epinephrine closure time. CONCLUSIONS: Unfractionated heparin leads to direct platelet activation and increases platelet aggregation in vitro. Unfractionated heparin causes prolongation of the collagen/adenosine diphosphate closure time in PFA-100 testing, possibly as a result of direct binding to von Willebrand factor in solution and interference with von Willebrand factor-glycoprotein Ib binding.

Adult↗

Effects of a fish oil supplement on platelet function, haemostatic variables and albuminuria in insulin-dependent diabetics.

A randomised trial of the effects of 15 gm per day of a fish oil supplement (MaxEPA) on blood lipids, haemostatic variables (including platelet function) and albuminuria was undertaken in 41 insulin dependent diabetics. Compared with the control group there was a significant reduction in thromboxane production by platelets stimulated by collagen in vitro in the group who took the fish oil supplement. The extent of platelet aggregation was not altered but the lag phase before aggregation was prolonged. There were also statistically significant increases in plasma LDL cholesterol, fibrinogen and clotting factor X in the group who took the fish oil supplement. No other significant differences were noted.

Administration, Oral↗

Increased platelet and coagulatory activity in peripheral atherosclerosis flow mediated platelet function is a sensitive and specific disease indicator.

The importance of flow in both athero- and thrombogenesis is well established. In peripheral arterial disease (PAD) atherosclerosis is disseminated, thrombosis risk high and systemic hemostatic derangement believed contributory. We studied platelet and coagulatory activity in PAD patients, thereby evaluating the clinical efficacy of Stagnation Point Flow Adhesio-Aggregometry (SPAA). SPAA provides real-time quantitative assessment of platelet adhesion and aggregation under convective, low-shear flow conditions. 62 nondiabetic PAD patients and 66 healthy volunteers were examined, whereby SPAA and conventional aggregometry were performed and circulating fibrinogen, fibrin monomer (FM), the fibrin degradation product D-Dimer and thrombin-antithrombin-complex (TAT) assessed. Conventional aggregometry detected no differences between patients and controls. SPAA-measured platelet function (p < 0.001), fibrinogen (p < 0.001), FM (p < 0.001), TAT (p < 0.02) and D-Dimer (p < 0.001) were significantly increased in patients and not effected by aspirin therapy. Respective sensitivity and specificity in detecting PAD was as follows: SPAA (96%/95%), fibrinogen (36%/92%), FM (46%/88%), TAT (40%/73%), D-Dimer (75%/80%). Increased platelet and coagulatory activity was verified in PAD, whereby flow-mediated platelet adhesion and aggregation proved the most sensitive and specific parameter. These findings indicate the usefulness of SPAA for delineating platelet-related disease mechanisms and evaluating therapeutic strategies to prevent platelet activation.

Antithrombin III↗

Enzymes that hydrolyze adenine nucleotides in diabetes and associated pathologies.

The activities of the enzymes NTPDase (E.C. 3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD39) and 5'-nucleotidase (E.C. 3.1.3.5, CD73) were analyzed in platelets of type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients. The results showed an increase in platelet NTPDase activity in type 2 diabetic (34% and 72%), hypertensive (32% and 70%) and type 2 diabetic/hypertensive patients (30% and 55%) when compared to control (P<.01) with ATP and ADP as substrate, respectively. 5'-Nucleotidase activity was elevated in the hypertensive (60%) and type 2 diabetic/hypertensive (53%) groups when compared to the control and type 2 diabetic group (P<.01). No differences in sensitivity to inhibitors was detected between the platelets of controls and type 2 diabetic/hypertensive patients. No effects on the enzyme activities were observed when pharmacological doses of propranolol, captopril, furosemide, chlorpropamide, acetylsalicylic acid and glibenclamide were administered. Furthermore, changes in platelet adhesiveness and reactivity were found in all groups tested. In conclusion, we may postulate that NTPDase and 5'-nucleotidase from platelets are altered in patients with type 2 diabetes and hypertension. Probably, such alterations are involved in compensatory physiological responses in these diseases and are related to other important mechanisms of thromboregulation.

5'-Nucleotidase↗

Influence of a 7-day treatment with Ginkgo biloba special extract EGb 761 on bleeding time and coagulation: a randomized, placebo-controlled, double-blind study in healthy volunteers.

During recent years, several case reports have been published in which the authors have voiced their suspicion of a causal relationship between hemorrhagic complications and the intake of Ginkgo biloba preparations. Therefore, a trial was conducted to investigate the influence of Ginkgo biloba special extract EGb 761 on hemostasiological parameters. Fifty healthy, male volunteers underwent 7 days of crossover treatment with 2 x 120 mg/day EGb 761 and placebo in randomized sequence. Between the two treatment phases, a washout-period of at least 3 weeks was inserted. The study's main outcome measures were bleeding time, coagulation parameters, platelet activity in response to various agonists and platelet morphology. The equivalence of the two treatments was analyzed by computing the 90% Fieller confidence intervals for the ratio between the means of the pre-post treatment differences for EGb 761 and placebo, respectively. Treatment safety was investigated by clinical laboratory and vital signs assessment and by adverse events monitoring. Among the 29 coagulation and bleeding parameters assessed, none showed any evidence of an inhibition of blood coagulation and platelet aggregation through EGb 761. Furthermore, the study did not reveal any evidence to substantiate a causal relationship between the administration of EGb 761 and hemorrhagic complications. As regards treatment tolerability, there were no interpretable differences between EGb 761 and placebo except for a slight increase of gastrointestinal complaints during administration of the herbal extract.

Adult↗

The effect of n-3 polyunsaturated fatty acids on lipids, haemostasis, neutrophil and monocyte chemotaxis in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is associated with an increased risk of coronary artery disease (CAD). There is some evidence that polyunsaturated fatty acids of the marine n-3 type (n-3 PUFA's) may offer protection against CAD. We have studied the effect of short-term dietary supplementation with n-3 PUFAs on lipids, haemostasis, neutrophil and monocyte chemotaxis in 10 patients with IDDM. The patients were given 4 g daily of n-3 PUFAs (fish oil) for 6 weeks and were investigated before and after the supplement. No significant effects on platelets or haemostasis were observed. High density lipoprotein (HDL)-cholesterol significantly increased, and triglycerides and the ratio of total cholesterol to HDL-cholesterol significantly decreased. Monocyte chemotaxis was unaltered, while neutrophil chemotaxis significantly increased after fish oil. The finding of an improvement in neutrophil chemotaxis after supplementation with n-3 PUFAs to patients with IDDM needs to be confirmed in future studies.

Adult↗

Clinical trial of a new disposable bleeding-time device.

A new disposable bleeding time device (Hemalet) was tested in 20 normal individuals and 11 patients with various bleeding disorders. The results were compared with those of Simplate II. The mean bleeding time for normal individuals was 5.4 +/- 1.5 (mean +/- 1 SD) minutes by Hemalet and 5.8 +/- 1.4 (mean +/- 1 SD) minutes by Simplate II, with good correlation between the results by the two devices (r = 0.81). The bleeding time in patients with various bleeding disorders were also comparably prolonged between the two devices. The new disposable bleeding time device with a disposable blade has quick release (penetration) into skin and retraction, and offers an alternative means of a bleeding time test.

Bleeding Time↗

The effects of hydroxyethyl starches on intracellular calcium in platelets.

UNLABELLED: Hydroxyethyl starch (HES) solutions impair platelet function. To determine whether this effect is achieved through interference of HES with intracellular activation processes, in which calcium is the key second messenger, we evaluated the agonist-induced increase of the cytoplasmic calcium concentration in the presence of HES of different molecular weights. Aliquots of citrated whole blood of 12 volunteers were incubated in vitr. with saline, HES 450 (molecular weight in kilodalton), HES 130, and HES 70, resulting in 20% hemodilution. An undiluted sample served as control. The samples were stained with Fluo-3 as the calcium-sensitive fluorescent probe with subsequent flow cytometric analysis. After determination of a baseline, platelets were activated with thrombin receptor activator peptide 6. Platelet activation with thrombin receptor activator peptide 6 resulted in a fast increase in fluorescence (approximately eightfold), representing intracellular calcium mobilization. None of the tested HES solutions exerted a statistically significant effect on the cytoplasmic calcium concentration compared with samples that were incubated with saline or that remained undiluted. These results indicate that the known inhibiting effect of HES on platelets is not achieved through interference with intracellular activation processes. IMPLICATIONS: Hydroxyethyl starch does not exert its known inhibitory effect on platelet function by interfering with intracellular activation processes.

Aniline Compounds↗