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PubMed1991

Hirudin for diagnostic purposes.

Hirudin serves as a versatile tool for the control of thrombin activity in hemostaseology. It may be added in excess to blood, plasma or test mixtures to prevent catalytic and nonenzymatic effects of thrombin. It may be used to quench thrombin activity upon extensive or limited action. Unlike heparin-antithrombin III complex which exclusively inhibits alpha-and gamma-thrombin, hirudin also inhibits meizothrombin, a precursor of alpha-thrombin. Thus, hirudin may be used for the detection of meizothrombin as well as for the prevention of its action in plasma samples. In conjunction with chromogenic substrates, hirudin may serve to discriminate between actions mediated by thrombin, its precursors, cofactors and effectors and actions of other enzyme systems. The principle of this hirudin application is exemplified for factor-V- and factor-VIII-dependent anticoagulant activity of protein C.

Amino Acid Sequence↗
PubMed1988

A new bleeding tendency due to hereditary hyper alpha 2-macroglobulinemia.

A heritable elevation in alpha 2-macroglobulin (alpha 2M) was identified in a 9-year-old girl with a severe bleeding tendency and activated partial thromboplastin time (APTT) prolonged to 49.1 sec (normal 27-38) as well as recalcification time prolonged to 438 sec (less than 180). The addition of her plasma to normal plasma made APTT prolong from 26.8 to 38.3 sec. The plasma alpha 2M levels in her relatives were checked, i.e., proband, her sister, mother, maternal grandmother, father, and paternal grandmother: Their levels were 406, 380, 352, 339, 166 and 236 mg/100 ml (140-285), respectively. Thus the patient's condition was thought to be an autosomal dominant disease, though her other relatives displayed no apparent clinical symptoms. Of significance was that a possible causal association between her elevated alpha 2M and her prolonged APTT was indicated. The activity of the alpha 2M, determined as trypsin-protein esterase, was 351 mg/100 ml (197%). The alpha 2M also demonstrated normal horizontal mobility to anti-alpha 2M plasma with a high precipitin arc (showing the difference of the protein concentration) by crossed immunoelectrophoresis, and a normal horizontal mobility of immunofixation electrophoresis. In addition, after analysis of testing done by SDS-polyacrylamide-gel-electrophoresis, we found no qualitative abnormality in the alpha 2M of the patient.

Blood Coagulation Disorders↗
PubMed1984

A comparison of the antithrombotic and haemorrhagic effects of low molecular weight heparin fractions: the influence of the method of preparation.

Bleeding is an important complication of heparin therapy. A number of low molecular weight heparin fractions produce less bleeding than standard heparin for an equivalent antithrombotic effect in experimental animals. Low molecular weight heparin fractions and fragments are produced by a number of different procedures but their relative effects on haemostasis and thrombosis have not been evaluated. We have compared the antithrombotic and haemorrhagic effects of two low molecular weight heparin fragments and of a heparinoid with porcine mucosa heparin and related these in vivo findings to the results of ex vivo tests of blood coagulation and in vitro tests of platelet vivo tests of blood coagulation and in vitro tests of platelet function. Haemorrhage was assessed using a rabbit ear bleeding model. The antithrombotic effects were assessed by measuring inhibition of a tissue thromboplastin-induced jugular vein thrombus and by inhibition of fibrin and platelet accumulation in an arterial-venous shunt. The ex vivo anticoagulant effects were assessed with the thrombin clotting time, activated partial thromboplastin time and anti-Xa assay, and the effect of these glycosaminoglycans on platelet function was assessed by measuring collagen-induced platelet aggregation. For a similar antithrombotic effect, standard heparin produced significantly more bleeding than the other 3 glycosaminoglycans. The antithrombotic effects of all 4 glycosaminoglycans occurred at similar levels of anti-Xa activity but there was no relationship between blood loss and the effects of these glycosaminoglycans on any of the other tests of blood coagulation. Standard heparin had a greater inhibitory effect on collagen-induced platelet aggregation than the low molecular weight glycosaminoglycans, supporting the possibility that the increased bleeding observed with heparin is related in part to its inhibitory effect on platelet function.

Animals↗
PubMed2000

Use of the PFA-100 apparatus to assess platelet function in patients undergoing PTCA during and after infusion of c7E3 Fab in the presence of other antiplatelet agents.

The PFA-100 (Dade) is a new functional whole blood analyzer, the accuracy and reliability of which have been evaluated in von Willebrand disease and during acetyl salicylate acid therapy. This new test has the advantages of rapidity and simplicity. It may be useful to monitor new antiplatelet agents, such as GPIIb/IIIa receptor antagonists. The objective of this study was to assess the PFA-100 in comparison with aggregometry and with the percentage of blockaded receptors GPIIb/IIIa during and after c7E3 Fab infusion in fifteen patients undergoing PTCA. Our results showed a change of closure time values from normal to abnormal within a small margin of flow cytometric values (60-75% of blockaded receptors), and moreover a variable platelet response to long-term low dose aspirin treatment in agreement with aggregometry. No influence with heparin was observed. In conclusion, this study shows that PFA-100 may be helpful in the decision making for additional antiaggregant therapy before PTCA or in monitoring long-term GPIIb/IIIa receptor antagonist treatment.

Abciximab↗
PubMed1989

An in vitro bleeding time test.

A new conical plastic device and method, the Platelet-Stat test, has been developed to measure in vitro bleeding time. Ten milliliters of citrated blood, collected by venipuncture, was used. The in vitro bleeding time test was validated by several criteria. Eight volunteers tested had a mean bleeding time of less than 1 minute. Different anticoagulants were evaluated, and the test performed optimally with citrate. Within-run precision had a mean time of 39 +/- 6.7 seconds with a coefficient of variation of 17%. An aspirin study was done on eight volunteers. Preaspirin in vitro bleeding time was less than 1 minute, whereas postaspirin times were more than 7 minutes at 18-24 hours. This test is a reproducible method of performing the bleeding time with greater precision than the in vivo test.

Anticoagulants↗
PubMed2000

Platelet dysfunction detected at high shear in patients with heart valve disease.

Whether patients with valvular heart disease have a defect of platelet function has been unclear. Despite evidence that these individuals have an abnormality detectable only under conditions of high shear stress, no methods have been widely available to adequately assess platelet function under such conditions. The Platelet Function Analyzer (PFA)-100 measures platelet function in a high shear environment and is well suited to the detection of platelet dysfunction in the clinical laboratory. The instrument records the time for platelets to occlude a membrane coated with collagen and either epinephrine (CEPI) or ADP (CADP). We studied the PFA-100 in 398 patients before open heart surgery; 308 for coronary artery bypass grafting (CABG) and 90 for aortic or mitral valve replacement (VR). Patients were classified as normal (CEPI < or = 153 s); 'aspirin effect' (CEPI > 153 s but CADP < or = 109 s) or abnormal (CEPI > 153 s and CADP > 109 s). In the CABG group, 41.2% were classified as normal, 43.2% as 'aspirin effect' and 15.6% as abnormal. In contrast, in patients undergoing VR, these values were 6.7, 11.1 and 82.4%, respectively. Patients with valvular disease had significantly longer closure times for both CEPI and CADP tests (P < 0.001). In addition, the valvular disease group had a significantly higher proportion of patients with markedly prolonged (> 150 s) closure times in the CADP cartridge (43.3 vs. 3.6%, respectively). Only one (0.3%) patient in the CABG group had non-closure (> 300 s) in the CADP test compared to seven (7.8%) in the valvular disease group. Three of six patients in the latter group bled excessively during surgery. We conclude that abnormal CADP closure is much more frequent among patients with aortic or mitral valve disease compared to those with coronary artery disease. This may reflect pre-existing high-shear damage to platelets that renders them refractory to subsequent shear activation and aggregation in the PFA-100 system. Further studies are needed to more precisely define the platelet defect in these patients. Markedly prolonged CADP closure in patients with valvular disease may indicate an increased likelihood of intra-operative bleeding, although an appropriately designed prospective study is needed to adequately address this hypothesis.

Adenosine Diphosphate↗
PubMed2002

Evaluation of platelet parameters on the ADVIA 120 as the quality indicator for stored platelets.

In order to investigate the possible use of platelet parameters on the ADVIA 120 hematologic analyzer as the routine quality control indicator for preparation and storage of platelets, platelet parameters, pH and CD62P expression were determined in stored platelet concentrates. Platelet component distribution width (PCDW) was decreased progressively on days 1 and 3 of storage for 5 days when compared with 0 day. PCDW correlated with CD62P expression on unstimulated platelets and the difference in CD62P expression following agonistic stimulation (a measure of functional reserve). Mean platelet component (MPC) was decreased on day 1 of storage. It did not however, show a progressive decrease over storage time and did not correlate with CD62P, although MPC has been known to be a useful screening test for platelet activation. Therefore, PCDW is considered to be a simple, convenient and cost-effective quality indicator for determining the viability and storage lesion of platelets for transfusion.

Blood Preservation↗
PubMed1978

Platelet function in hyperbetalipoproteinemia.

Individuals with familial hyperbetalipoproteinemia are at increased risk of premature atherosclerosis and thrombosis. Although there is controversy whether platelet survival is shortened or normal in this disease, several in vitro tests of platelet function are abnormal including a decreased threshold concentration for stimulation of aggregation by ADP, epinephrine and collagen and increased release of nucleotides to the same agents. These functional changes are accompanied by an increase of cholesterol to phospholipid ratio in the platelet membrane and in low density lipoprotein in individuals with type IIa hyperlipoproteinemia. Clofibrate and halofenate reverse some of the abnormalities in vitro and the former drug, when administered for 6 weeks to patients with type IIa hyperlipoproteinemia decreases platelet sensitivity to ADP and epinephrine. The platelet hypersensitivity to aggregating agents can be reproduced in vitro by increasing the cholesterol to phospholipid rather in normal platelets. These artificially hypersensitive platelets can be returned to normal by halofenate in vitro. Incorporation of cholesterol into platelet membranes increases the basal level of the membrane associated enzyme adenylate cyclase. However, the enzyme no longer responds to stimulation by prostaglandin E1, and this is associated with relative resistance of the platelet to inhibition by this pharmacologic agent. These functional alterations produced by cholesterol enrichment of platelet membranes occur is parallel with an increase in platelet membrane microviscosity suggesting that the more rigid membrane can alter the behavior of membrane associated enzymes and receptors. A correlation appears to exist between the ability of certain drugs to induce phase separation in model membranes and the potency in inhibitory platelet aggregation.

Adenosine Diphosphate↗
PubMed2006

Central laboratory and point of care assessment of perioperative hemostasis.

PURPOSE: To review laboratory investigations required for the diagnosis of a constitutional or acquired defect of hemostasis before surgery, or during the perioperative period in context of rapid evolution and possible therapeutic adjustment. METHODS: A review of the literature. PRINCIPAL FINDINGS: Systematic preoperative screening is poorly efficient. It should be restricted to patients selected on clinical history and physical examination. Intra- and postoperative investigation is oriented by the clinical circumstances. In most instances, laboratory investigation is organized as hierarchical steps, the first one including a platelet count, a prothrombin time and activated partial thromboplastin time. According to the results of these assays and to the clinical circumstances, further tests specific of primary hemostasis, coagulation or fibrinolysis are performed. Point of care (POC) monitoring has been developed more recently for the investigation of a perioperative bleeding. Several tests examine platelet functions, others measure whole blood activated partial thromboplastin time or prothrombin time and some explore global hemostasis, allowing the detection of excessive fibrinolysis. Point of care testing provides a rapid and valuable answer but, if one accepts the monitoring of unfractionated heparin during extracorporeal circulation, most assays have not been properly validated. CONCLUSION: The investigation of hemostasis at the central laboratory and POC testing have distinct objectives. The utility of the former for the diagnosis and the adjustment of therapeutics have been well demonstrated. In contrast, the experience with POC testing is relatively recent, and its utility for patient management remains to be demonstrated in proper clinical trials.

Blood Coagulation Disorders↗
PubMed1989

Platelet hyperreactivity and inefficient spontaneous thrombolysis in patients at high risk from an acute coronary event.

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.

Adult↗
PubMedDate not supplied

[Antithrombin III and alpha-2-antiplasmin activities compared with other hemostasis parameters in uncomplicated diabetes mellitus, type 2].

In 40 patients with diabetes mellitus type II without clinical signs of any organ complications and in the respective control group the following indices of hemostasis were assessed: 1) activity of AI-III, 2) activity of alfa-2-AP, 3) fibrinogen, 4) time of fibrinolysis, 5) platelets count, adhesiveness and spontaneous aggregation, 6) kaolin-cephalin and profil stipven-cephalin plasma times. All these indices were normal in uncomplicated diabetes mellitus with the expectation of platelets activity. Stimulation of platelets activity and increase of corresponding parameters appears in diabetes mellitus type II before any other symptoms of angiopathy.

Adult↗
PubMed1991

Predictive value of coagulation testing in cardiopulmonary bypass surgery.

Severe postoperative bleeding in cardiopulmonary bypass surgery is still a major problem. One hundred and seven patients undergoing uncomplicated coronary artery bypass grafting (CABG) were studied. Two groups were formed according to the patients' 24 hours postoperative mediastinal blood loss. Patients in group I (n = 70) had blood loss less than 1000 ml and patients in group II (n = 33) had blood loss over 1000 ml. Between the two groups, there were no differences in age, male-female ratio, number of grafts, by pass-time or heparin-protamine dose. Coagulation testing: APTT (Activated Partial Thromboplastin Time), PT (Prothrombin Time), fibrinogen, D-dim. (D-dimers), bleeding time, MPV (mean platelet volume) and platelet count, was done preoperatively (T0), immediately postoperatively (T1), 6 hours postoperatively (T2). We were especially interested in the predictive value of hemostatic parameters at T0 and T1. We found a slight but statistically negative correlation between the platelet count (T0 and T1) and the postoperative blood loss.

Blood Coagulation Tests↗
PubMed1984

Inherited fibrinolytic disorder due to an enhanced plasminogen activator level.

A family with "in vitro" increased red-cell fall out from the blood clot was studied. One member of the family (JVM) had a clinical history of hemorrhages after minor trauma or dental extractions. Routine coagulation and platelet function were normal except for the fibrinogen level which was slightly low in several members. The antigenic as well as functional evaluation of factor XIII was within normal limits. No factor XIII inhibitors were present. An increase in the clot permeability index was observed in most family members. The study of the fibrinolytic system showed an enhanced lysis of euglobulins, a normal plasminogen value, normal level of fibrin/ogen degradation products, normal fibrinolytic inhibitors, and an increase in the activity of the plasminogen activator. The activity of this activator was inhibited by an antiserum against tissue-type plasminogen activator. The t-pA inhibitor was in the normal range. It is concluded that the family studied in this paper shows familial alteration in the fibrinolytic system due to an excess of plasminogen activator immunologically related to that in human tissue.

Adult↗
PubMedDate not supplied

Platelet abnormalities in idiopathic myelofibrosis: functional, biochemical and immunomorphological correlations.

BACKGROUND: An extensive study of platelet function was performed on 18 consecutive patients affected by idiopathic myelofibrosis (IM). MATERIALS AND METHODS: Clinical hematological and morphofunctional parameters were studied in IM patients and control subjects. Platelet tests, ultrastructural data, immunocytochemical von Willebrand factor detection, freeze fracturing results and free cytosolic calcium level were evaluated. RESULTS: Bleeding time was frequently found to be prolonged, but it never reached levels which could give any cause for concern. Aggregation by ADP, collagen and epinephrine was always altered, sometimes profoundly; on the contrary, agglutination by ristocetin was almost always normal, albeit occasionally increased. Plasma beta-TG and PF4 levels were found to be elevated in 11 and 12 patients, respectively. This indicated an abnormal release from platelet alpha-granules. Depletion of alpha-granules was also confirmed by the intraplatelet von Willebrand factor (vWF) labelling with colloidal gold particles bound to polyclonal antibodies against human vWF. In fact: 1) the number of positive alpha-granules/microm2 and per single platelet was reduced; 2) the intensity of the immunocytochemical reaction for single positive alpha-granules and for each platelet was significantly reduced. Freeze-fracturing studies showed an increase in the number of intra-membrane particles (IMP) on the P face of the platelet membrane with respect to normal platelets preincubated with ADP. However, no differences in their distribution or diameter were observed. High concentrations of free cytosolic calcium were always found and Ca++ ATPase activity was increased. Conversely, Na+/K+ ATPase activity was always reduced. CONCLUSIONS: We can hypothesize that the platelet membrane is altered in IM, resulting in facilitated activation, even by subliminal stimuli, and that this continuous platelet activation ultimately leads to alpha-granule depletion.

Adenosine Diphosphate↗