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Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Most of our understanding of platelets is based on their function in hemostasis, as it relates to quantitative and qualitative bleeding disorders. In hemostasis, platelets perform two major functions. First, because of their ability to adhere and aggregate at sites of endothelial injury, they facilitate the initial arrest of bleeding, also called primary hemostasis. Second, on ‘‘activation’’ at the site of vessel injury, they provide the surfaces onto which the factors of the coagulation system can be bound, thus keeping clot formation localized. The physiology and biochemistry of platelets has been the subject of extensive reviews [1–4] and will not be discussed in this context. Although the pathophysiology of the role of platelets in bleeding disorders is reasonably well understood, less is known about the involvement of platelets in thrombosis and atherosclerosis, though more patients have the latter than have bleeding problems. ‘‘Discovery of such abnormalities is the challenge of the future in platelet research’’ [2]. The role of platelets in the pathogenesis of arterial thrombosis in particular is relatively well known. Most arterial thrombotic events reside in clot formation at sites of atherosclerotic lesions, though other vascular diseases can also be involved. Ruptures at the base of the atherosclerotic plaques appear to precipitate thrombus formation [5,6]. As in normal hemostasis, platelets will adhere to exposed collagen fibers, aggregate, and initiate, together with tissue factor (TF), the activation of the coagulation system [7]. This leads to thrombus formation with temporary or permanent vascular occlusion. Atherosclerosis is the most prevalent reason for the development of acute myocardial infarction (AMI)

The risk of thromboembolic complications with the use of second and third generation oral contraceptives is minimal and probably related to underlying congenital or acquired thrombophilic states. Estrogen dose-dependency leads to increased thrombin generation and increased plasmin generation. There is no convincing evidence that the balance between clotting and fibrinolysis is disturbed. The risk of venous thromboembolism with pregnancy is greater than with oral contraceptives. Hormone replacement therapy is safe for healthy women, and the benefits far outweigh the potential risks.

Sepsis and its associated complications of disseminated intravascular coagulation (DIC) and multiple organ dysfunction syndrome (MODS) continue to be a major cause of morbidity and mortality. Improved detection of all forms of DIC is essential to assure earlier diagnosis. Studies already indicate that the therapeutic use of antithrombin (AT) concentrate may produce a more positive outcome for sepsis-associated DIC. If DIC could be identified earlier and AT concentrate could then be given earlier in the sepsis continuum, study results for the use of AT concentrate in humans might reveal a statistically significant difference versus placebo, and the efficacy of AT concentrate for this syndrome is more likely to be proved. Fixed-bolus doses of AT concentrate based on body weight are currently preferred, but improved, user-friendly assays for plasma AT levels would permit more rapid turnaround time for AT results and could help fine-tune the use of AT concentrate to the specific needs of each patient. Clinical trials involving the therapeutic use of AT concentrate in sepsis should continue, and it can be hoped that their design will reflect the concepts and conclusions offered by this panel of investigators.

Antithrombin (AT) is a single-chain glycoprotein in plasma and belongs to the family of the serpins. It is synthesized in liver parenchymal cells, and its plasma concentration is between 112-140 mg/L. AT is a unique inhibitor of the clotting system and neutralizes most of the enzymes generated during activation of the clotting cascade, especially thrombin, factors Xa and IXa. Equimolar, irreversible complexes are formed between AT and the enzymes. The interaction between AT and the activated clotting factors is at least 1,000-fold increased in the presence of heparins. Heparins bind to multiple sites of the AT molecule resulting in a steric reconfiguration. Heparins contain a specific pentasaccharide unit which is the minimum requirement for AT binding. The glycosaminoglycan (GAG) heparan sulfate found on endothelial cell surfaces also contains this pentasaccharide and can thus "activate" AT. It is believed that much of the physiological inactivation of enzymes by AT occurs on the endothelium, mediated by heparan sulfate. The binding of AT to the GAGs also releases prostacyclin which possesses strong antiinflammatory properties. Deficiencies of AT are inherited or acquired. Only acquired defects due to increased consumption are discussed, most notably AT in DIC, especially DIC in sepsis. During acute DIC, clotting factors and inhibitors are consumed faster than they can be reproduced. This consumption of AT is of great significance in DIC and sepsis, and plasma AT levels predict outcome. AT levels drop early in sepsis and laboratory signs of DIC can already be found in patients with SIRS and early sepsis. The important role of AT in DIC and sepsis is the basis for considering antithrombin concentrates as an additional therapeutic modality.

Background.During surgery, the balance between thrombosis and fibrinolysis is altered. Methods reported to increase fibrinolysis, such as compression devices, may reduce venous thrombosis. However, there are no prospective studies comparing methods and the effect on fibrinolysis.Materials and methods.In a prospective study, general surgical patients were randomized to either sequential compression devices (Group 1) or subcutaneous heparin (Group 2), and fibrinolysis factors were measured in order to determine the effect on the fibrinolysis system. Blood samples were drawn at a similar time of the day with the tourniquet off. Specifically, t-PA antigen, plasminogen activator inhibitor-1 (PAI-1), and D-dimer were measured preoperatively (preop) and on Postoperative Days (POD) 1 and 7 by the ELISA method. Fibrinolysis factors were reported as the mean ±SD and as percentage change from preoperative values. Noninvasive vascular studies were performed preop, and on POD 1, 7, and 30, by an examination of the infrainguinal venous system and external iliac veins in bilateral lower extremities. Nonambulatory patients were excluded from the study and DVT prophylaxis methods were initiated at surgery and used through POD 2.Results.For the 136 patients in the study, there were no differences in clinical characteristics such as age, surgical time (all >60 min), anesthesia type (general or spinal), type of surgical procedure, or other risk factors for DVT. Two DVTs occurred at POD 1 and 30 (both Group 2), and one pulmonary embolism in each group (POD 7 for Group 1; POD 1 for Group 2). For subjects without thrombosis, D-dimer changes were parallel for both groups, increasing through POD 7. Similarly, t-PA antigen levels rose from baseline on POD 1 in both groups, with a return toward baseline by POD 7. The PAI-1 levels increased on POD 1 in both groups, but severalfold more in Group 1 (compression devices). The elevation in PAI-1 decreased by 50% in Group 1 by POD 7, while values returned to normal in Group 2. These changes were not significant using the Mann-Whitney test. Only three patients had thrombotic episodes so that data on changes in fibrinolysis factors are difficult to compare with the larger group.Conclusions.This is the first report of a prospective, randomized comparison of fibrinolysis factors using sequential compression devices in comparison to low dose unfractionated heparin in general surgical patients, and comparing postoperative values to preop. Both groups showed an enhanced fibrinolysis by elevation in t-PA antigen and D-dimer on POD 1, as expected when fibrinolysis occurs. While PAI-1 and t-PA work in parallel, the marked elevation of PAI-1 on POD 1 (although only slightly above reference values) and continuing into POD 7 for subjects using compression devices requires further inquiry. The elevation of PAI-1 in the face of elevated t-PA and D-dimer has been reported, but the comparison between patients using sequential compression devices and mini-dose heparin has not been reported. The reason for the elevation requires additional study into other influences on the synthesis, secretion, and/or function of PAI-1 that do not affect t-PA.

Haematological changes in the septic patient are, primarily, neutropenia or neutrophilia, thrombocytopenia and disseminated intravascular coagulation (DIC). Thrombocytopenia frequently arises from DIC although inhibition of thrombopoiesis or immunological platelet damage also occur. DIC contributes to bleeding and microvascular thrombosis, leading to multiple organ failure. Tissue factor release, primarily mediated by tumour necrosis factor, activates the clotting system; fibrinolysis is initially activated, but later becomes inhibited by the release of plasminogen-activator inhibitor (PAI-1), further fostering multiple organ failure. Most septic patients have compensated, chronic DIC, detectable by assays of molecular markers; the earliest signs are already found during the systemic inflammatory response syndrome. Compensated DIC later becomes decompensated with rapid consumption of factors including inhibitors such as antithrombin III (AT III) and proteins C and S. AT III concentrations of < 60-70% of the normal values predict outcome. Management of DIC must address the underlying disease, interrupt the activated haemostasis system and replace consumed coagulation constituents. Interruption of haemostasis with heparin may be attempted, but bleeding may worsen. Administration of a natural anticoagulant, such as AT III, may arrest clotting without concomitant risk of bleeding. In several animal models of DIC, AT III concentrates shortened the duration of DIC and reduced multiple organ failure and mortality. Similar benefits have been reported in early studies of patients with DIC, especially in the absence of sepsis. Studies are under way to determine whether outcome will improve if patients with sepsis are treated before the development of shock and plasma AT III concentrations are maintained at 100-150% of normal.

Heart failure is associated with a hypercoagulable state. A single-center, randomized, double-blind, placebo-controlled trial was performed to test the hypothesis that warfarin will modify a hypercoagulable state in heart failure. This study included 76 patients with heart failure. At baseline, patients had evidence for a hypercoagulable state with elevated plasma levels of thrombin/antithrombin III (TAT) complexes (3.4 +/- 2.0 ng/ml), prothrombin fragment F1 + 2 (1.5 +/- 0.9 nmol/L), and D-dimers (630 +/- 401 ng/ml). Warfarin therapy (international normalized ratio [INR] 2.7 +/- 1.3) significantly decreased plasma levels of TAT complexes (p < 0.002), F1 + 2 (p < 0.001), and D-dimers (p < 0.001) when compared with baseline values at 1, 2, and 3 months of therapy. In contrast, patients receiving placebo had persistent elevation of TAT complexes (p = not significant [NS]), F1 + 2 (p = NS), and D-dimers (p = NS) during follow-up at 1, 2, and 3 months. The two treatment groups followed different trends over time for all three markers (p < 0.001). The effect of low-intensity warfarin (INR 1.3 +/- 0.08) versus moderate-intensity warfarin (INR 2.3 +/- 1.1 ) on markers of hypercoagulability was evaluated in 14 patients. When compared with baseline, low-intensity warfarin administration decreased plasma levels of TAT complexes (p = NS), F1 + 2 (p = 0.05), and D-dimers (p = 0.04). In these patients F1 + 2 was further reduced with moderate-intensity warfarin (p < 0.001). Our findings suggest that a hypercoagulable state in heart failure can be modified by warfarin therapy.

Objective: To determine the effects of elevated endogenous E 2 levels on in vitro platelet function in patients undergoing controlled ovarian hyperstimulation (COH). Design: Women with normal ovulatory cycles and patients undergoing COH on cycle day 3 and near ovulation (preovulatory follicles were at least 16 mm in diameter) were studied. Serum E 2 , Thrombostat 4000, (V. d. Goltz, Seeon, Germany), von Willebrand factor antigen (vWF-Ag), and platelet aggregation and adenosine triphosphate (ATP) release to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA) were measured. Setting: University-based outpatient infertility clinic. Patient(s): Twenty-two consenting infertile women undergoing COH cycles and 14 women with documented ovulatory cycles. Main Outcome Measure(s): Whole blood platelet aggregation with ADP, COL, AA, and Thrombostat 4000. Result(s): Estradiol levels rose significantly at peak times (P = 0.011). No changes were noted in in vitro platelet function measured by the Thrombostat 4000 and by whole blood platelet aggregation with ADP and AA and in ATP release with ADP, COL, or AA. Aggregation with collagen was increased because of likely elevations in vWF-Ag levels. Conclusion(s): No significant changes in in vitro platelet function were noted in 19 women undergoing COH with E 2 levels two to three times that observed in oral contraceptive or hormone replacement therapy users, suggesting no increased risk for arterial thromboembolism.

SummaryArdeparin sodium (Normiflo®, Wyeth-Ayerst) is a low molecular weight heparin undergoing clinical evaluation as an antithrombotic agent. The objective of this study was to evaluate the absolute and comparative bioavailability of ardeparin following subcutaneous administration of three different formulations [two formulations of ardeparin at 10,000 anti-factor Xa (aXa) U/ml, but with different preservatives, and a 20,000 aXa U/ml formulation]. The study was conducted using a randomized 4-period crossover design (three subcutaneous treatments and one intravenous treatment) in 24 healthy subjects, and the pharmacokinetics of ardeparin were characterized by plasma anti-factor Ila (alia) and anti-factor Xa (aXa) activities. The mean absolute bioavailability of ardeparin based on alia activity ranged from 62% to 64% and the mean absolute bioavailability based on aXa activity ranged from 88% to 97%. Based on bioequivalence testing criteria, the three ardeparin formulations were bioequivalent.

Protein S can be determined by functional or immunological assays. Electroimmunodiffusion (EID) or enzyme immunoassays (enzyme-linked immunosorbent assay; ELISA) are the commonly employed techniques for measuring protein S and C4b-binding protein (C4b- BP) immunologically. Procedures for these assays are time-consuming and labor-intensive. The introduction of microlatex immunoassays (LIATEST system; Diagnos tica Stago, Asnieres-Sur-Seine, France) has provided an alternative for rapid and reliable immunological determi nation. We have placed the microlatex immunoassay for total and free protein S (TPS, FPS) and C4b-BP, using the light-scattering mode, on the Automated Coagulation Laboratory (ACL) 300 Plus (Instrumentation Laboratory, Lexington, MA, U.S.A.). We also placed a functional activity assay of protein S (STACLOT protein S; Amer ican Bioproducts, Parsippany, NJ, U.S.A.) on the ACL 300 Plus. The performance characteristics for the assays yielded a within-run coefficient of variance (CV) of 2.5- 4.6% ( n = 13) for TPS, 4.0-4.8% ( n = 13) for FPS, 1.9- 3.0% ( n = 11) for C4b-BP, and 2.3-5.9% for protein S activity. The interrun CV was 2.1-5.7% ( n = 24), 3.7- 7.0% ( n = 12), 2.6-7.0% ( n = 16), and 4.0-8.4% ( n = 27), respectively. Analytical recovery was 94-109, 97-100, 91-103, and 99-103%, respectively. The normal ranges determined on plasmas from 30 healthy individuals were 113 ± 37 (mean ± 2 SD) for TPS, 106 ± 35 for FSP, 111 ± 22 for C4b-BP, and 107 ± 34 for protein S activity. The results for the microlatex immunoassay and either the EID or the ELISA methods showed excellent correla tions for FPS and C4b-BP; the correlations between LIATEST and either EID or ELISA for TPS were also relatively high. The functional activity of protein S cor related well with FPS. Microlatex immunoassays, using the light-scattering mode for TPS, FPS, or C4b-BP, and the functional assay of protein S can be adapted on the ACL 300 Plus system with a high accuracy and reproduc ibility and with considerable time saving.

We investigated the effect of four thrombo plastins with different International Sensitivity Index (ISI) values on prothrombin times (PTs), expressed as International Normalized Ratios (INRs), of 30 normal plasmas, 30 patients on stable oral anticoagulant (OA) therapy, and 30 patients with prolonged PTs for reasons other than OA therapy. Normal ranges became signifi cantly longer when three thromboplastins of rabbit brain origin were used—the lower the ISI, the wider the normal range. The human placenta-derived thromboplastin had a range similar to the values obtained with the most insen sitive rabbit reagent. Significantly different INR values were noted when normal plasmas and plasmas of patients with prolonged PTs not due to OA therapy were tested. This was not the case when specimens from patients on OAs were tested. When PT values from 212 consecutive ly selected patients were tested with the least sensitive rabbit reagent (ISI 2.477) and the most sensitive human placenta reagent (ISI 1.06), significantly more "abnor mal" PTs were identified with the high-sensitivity throm boplastin. The data indicate that special attention has to be paid to "normal" ranges when thromboplastins with low ISI replace less sensitive reagents, that INR should not be used to express PT in patients not on stable OA therapy, and that with the routine use of highly sensitive reagents (low ISI) more abnormal patients are identified.

Ardeparin is a low molecular weight heparin currently being evaluated as an antithrombotic agent. The objective of this investigation was to assess the effects of dose on the pharmacokinetics of ardeparin after subcutaneous administration. Eighteen healthy subjects received doses of 30 U/kg, 60 U/kg, and 100 U/kg antifactor Xa (aXa) of ardeparin by subcutaneous injection. Plasma antifactor IIa (aIIa) activity levels after the 30- and 60-U/kg doses of ardeparin were too low to reliably characterize the disposition of the drug. However, the pharmacokinetics of ardeparin could be characterized by using pharmacodynamic measurements of plasma aXa activity. The rate of absorption of ardeparin after subcutaneous administration did not change with increasing dose. The volume of distribution (Vd) of ardeparin was small, reflecting minimal distribution outside the intravascular space, and was independent of dose. The total clearance of ardeparin, however, decreased with increasing dose, and half-life (t1/2) was prolonged at the higher doses. Within the observed dose range, a doubling of the ardeparin dose resulted in an area under the plasma aXa activity-versus-time curve (AUC) that was approximately 25% greater than expected on the basis of linear disposition. The differences in AUC and clearance between the three doses suggest that the mechanism of elimination of ardeparin is saturable.

Anabolic-androgenic steroid abuse has recently been linked with acute vascular events in athletes. To date, the relationship between steroid abuse and the potential for cardiovascular disease has been considered almost exclusively in terms of lipid metabolism. However, recent reports of thrombosis in androgen abusing athletes with no evidence of atherosclerosis suggests the hypothesis that thrombosis risk in such athletes could be mediated through androgen induced abnormalities of coagulation. To determine if anabolic-androgenic steroid abuse in weight lifters is associated with an activation of the hemostatic system we studied forty-nine weight lifters recruited through advertisements. History of androgen use or abstinence was confirmed via urine assays. Plasma was assayed for clotting and fibrinolytic activity by measuring thrombin/antithrombin complexes (TAT), prothrombin fragment 1 + 1 (F1 + 2), and D-dimers (D-di); markers of the endothelial based fibrinolytic components were assayed by measuring tissue plasminogen activator antigen (t-PA Ag) and its inhibitor (PAI-1); finally, the activity of antithrombin III, protein C, and protein S were measured. Abnormally high concentrations of TAT complexes were noted in 16% of our confirmed steroid using weight lifters compared to 6% of our confirmed nonusers (P = .01). Steroid users also demonstrated abnormally high concentrations of F1 + 2 and D-dimers when compared to nonusers (44 vs. 24%, P < .001, and 9 vs. 0%, respectively). Non-steroid users were more likely to have elevated levels of t-PA Ag and PAI-1 than our steroid using weight lifters (both P < .001). The activities of antithrombin III and protein S were more likely to be higher in users compared to nonusers (22 vs. 6%, P = .005; 19 vs. 0%, respectively). Some anabolic-androgenic steroid using weight lifters have an accelerated activation of their hemostatic system as evidence by increased generation of both thrombin and plasmin. These changes could reflect a thrombotic diatheses that may contribute to vascular occlusion reported in young athletes using these drugs. The predictive value of these coagulation abnormalities in terms of risk of thrombosis to individual steroid using weight lifters or the population as a whole remains to be studied.

The performance characteristics of an in vitro bleeding time device — Thrombostat 4000 were evaluated and compared with the Simplate bleeding time in healthy individuals and patients with disorders of primary hemastasis. Reference ranges were established using 30 normal volunteers. Although there were variations between different filter batches, reproducibility was good within a single batch. There were no differences between the two channels of the instrument and between male and female subjects. Hematocrit correlated negatively with the initial flow (IF) and IF correlated positively with closure time (T) and bleeding volume (V). Aspirin could be detected only when the traditional addition of ADP was replaced with CaCl 2 . Both, closure time (T) or bleeding volume (V) were more sensitive than Simplate bleeding time and T was more sensitive than V in detecting patients with disorders of primary hemostasis. We conclude that the Thrombostat 4000 is a reproducible, reliable, sensitive and easy to use instrument. It is superior to the traditional in vivo bleeding times for investigations of disorders of primary hemostasis (screening, diagnosis, monitoring, etc.).

73 healthy women (29 controls, 25 using OCs, and 19 using Norplant) were selected from the clinic population at North Oakland Medical Center for inclusion in this study after obtaining informed consent. Age, race, height, weight, blood pressure, and cigarette smoking were recorded for each subject. 12 patients were on monophasic OCs while 13 were on triphasic preparations. Both hormonal contraceptive groups had used their particular contraceptive for at least 3 months prior to blood drawing. Platelet tests were performed within 2 hours of sample collection: platelet counts (PLC) and mean platelet volume (MPV) were determined on an Automated Platelet Counter (Baker 810 Platelet Analyzer). Whole blood aggregation was performed on a platelet aggregometer (Chrono-Log, Model 550) using both ADP (ADP, 5 mM) and collagen (COLL, 2 mcg/ml) as inducing agents. Demographic differences were not significant (p 0.05) among the 3 treatment groups, whose average age was 25.3-25.8 years old. Furthermore, no significant differences (p 0.05) in platelet function were detected among controls or subjects receiving either oral contraceptives or Norplant, compared to control patients. The mean platelet counts (X 10/9/L) were 223 for OC users, 231 for Norplant users, and 232 for controls. The respective platelet aggregation (ADP, ohms) values were 12.5, 18.0, and 19.2 as well as (COLL, ohms) 35.6, 40.7, and 39.0. These results demonstrated that there is no evidence for altered platelet function, with the testing methods employed, in women using either Norplant or combination low dose oral contraceptives. To date, several studies have examined this issue, with contradictory reports about the effects of hormonal contraceptives in platelet function. After controlling for differences between various steroid preparations and other such confounding variables, some of these conflicting conclusions could be the result of a lack of uniformity among the methods used to evaluate platelet aggregation. The ability to draw conclusions regarding altered in vivo thrombotic potential from these studies is thus questionable.

We describe a 10-year experience with a con genital platelet abnormality characterized by hyperaggre gability with ADP and/or epinephrine. The syndrome has so far been identified in >200 patients and their families. Clinical symptoms are transient or permanent arterial oc clusions, thrombotic in nature, affecting especially the coronary and cerebral vasculature, including ophthalmic vessels. The patients have no identifiable risk factors and are usually young (5-45 years), and other hypercoagula ble states are excluded. In many cases the vascular acci dent is precipitated by severe stressful events. Occasion ally the syndrome is found in patients with recurrent ve nous thromboembolism while they are on optimal oral anticoagulant therapy. In the laboratory the patients' platelets are hyperaggregable with decreasing ADP and/ or epinephrine concentrations added to platelet-rich plasma. Two forms are identified: Type I is marked by hyperaggregability with ADP and epinephrine, while Type II evidences hyperaggregability only with epineph rine. No abnormalities are found when other aggregation stimulators are used. By electron microscopy the plate lets are more readily activated by surface contact, with a greater tendency toward aggregate formation. Plasma lev els of platelet release proteins, platelet factor 4, and β-thromboglobulin are not elevated. The syndrome is treated with low-dose aspirin, which reverses the hyper aggregability and improves clinical symptoms. We hy pothesize that in vivo adrenaline release leads to in creased platelet clumping, which can transiently or per manently occlude small vessels. It is conceivable that altered surface receptors on the platelets might be respon sible for this congenital problem.

Disturbed liver parenchymal cell function adversely impacts on the hemostasis system, the extent of which correlates with the degree of liver disease. Because liver parenchymal cells synthesize most factors of the clotting and the fibrinolytic systems, levels of these procoagulant and anticoagulant as well as profibrinolytic and antifibrinolytic factors will decrease in plasma. These changes may be minor in patients with mild liver disease but are severe in patients with cirrhosis. Thrombocytopenia and thrombocytopathy usually complicate the clinical presentation, and systemic activation of the fibrinolytic system is always seen in cirrhotic individuals. Whether this fibrinolytic activation is primary or secondary in response to DIC is controversial. Some of the laboratory findings in DIC may be a reflection of decreased hepatic clearance of activation products by the reticuloendothelial system of the diseased liver. In patients with vitamin K deficiency or in those receiving oral anticoagulants, only the vitamin K-dependent procoagulants and anticoagulants are altered; all other parameters remain in the normal range. Laboratory changes associated with various surgical interventions involving the liver depend on the underlying pathology. Severe hemorrhages are encountered during orthotopic liver transplantation. During the anhepatic phase and during the reperfusion phase, there is a major activation of the fibrinolytic system. It is unclear whether this fibrinolytic response is primary or secondary. The use of antifibrinolytic agents has markedly reduced the clinical bleeding and, thus, the requirement for blood and blood products. Bleeding associated with partial liver resection is usually mechanical in nature, but peritoneovenous shunt operations can result in DIC. Ascites fluid is the trigger. The injection of thrombin containing sclerosants can also activate the hemostasis system in vivo, although, generally, no clinically detectable adverse reactions are noted.

This study was undertaken to establish a correlation between prothrombin activation fragment F 1 + 2 and one-stage prothrombin time (PT) ratios in patients receiving oral anticoagulant therapy. One hundred consecutive patients on warfarin treatment were utilized for this study. The patients had received warfarin for not less than four days prior to entry into the study. F 1 + 2 levels and PT ratios were found to be 0.28 +/- 0.24 nM/L (mean +/- SD) ranging from 0.01 to 1.5 nM/L and 1.62 +/- 0.46 (mean +/- SD) ranging from 0.97 to 3.11, respectively. Most patients on oral anticoagulants with PT ratios between 1.2 - 1.7 exhibited decreased concentrations of F 1 + 2. Normal control values of F 1 + 2 were established for this study in 40 healthy individuals; they were 0.40 +/- 0.23 nM/L (median +/- SD) ranging from 0.11 to 1.19 nM/L. Mean plasma levels of F 1 + 2 were significantly lower in the anticoagulated patients as compared to the healthy controls (t = 2.377, p < 0.05). The relationship between F 1 + 2 levels and PT ratios in the 100 anticoagulated patients was analyzed by linear regression. No significant correlation (r = -0.208) was found between F 1 + 2 levels and PT ratios. It is concluded that the degree of reduction in F 1 + 2 levels is not proportional to the intensity of therapy reflected by the PT ratios in anticoagulated patients.

A group of 56 women on OCs and a control group of 47 women not taking OCs were studied. The mean time of OC use was 4.2 + or - 4.1 years. 38 patients took triphasic pills (Orthonovum 777 [35 mcg ethinyl estradiol] or Triphasal [30 40 30 mcg ethinyl estradiol]) 11 took low-dose estrogen combination products and 7 took Ovral (50 mcg ethinyl estradiol). Blood plasmas were separated for batch analysis. The effect of these OCs was tested on 2 newer molecular markers of in vivo clotting activation using commercially available kits: Thrombin-Antithrombin III (TAT) complexes and Prothrombin Fragment 1+2 (F 1+2). TAT complexes form when thrombin is generated in vivo and bound to antithrombin III. Elevated plasma levels suggest increased in vivo thrombin generation. F 1+2 represents the NH2 terminal part of the prothrombin molecules that does not contain the thrombin moiety. Elevated plasma levels suggest the formation of thrombin. The normal levels of TAT complexes range from 1.0 to 5.0 mcg/L with no sex differences. Normal F 1+2 levels range from 0.36 to 0.95 nMol/L. Females have higher values (0.64 + or - 0.19) than males (0.46 + or - 0.15). While the mean age between the 2 study groups was significantly different (p 44 years). The control group was significantly older (36 + or - 12) than the OC user group (25 + or - 5) yet their F 1+2 levels were not different. OCs do not seem to affect the hemostasis system directly however those women who develop thromboembolic complications while on oral contraceptives might have congenital or acquired hemostasis defects predisposing them to thromboembolism.

OBJECTIVE: The effects of postmenopausal hormone replacement therapy on thrombosis remain controversial. We tested the hypothesis that estrogen or progesterone has no significant effect on thrombosis by means of newly developed markers of blood clotting, specifically prothrombin fragment 1 + 2, a marker of factor Xa generation, and thrombin-antithrombin III complex, a marker of thrombin generation. STUDY DESIGN: A prospective study that included 106 women, 68 postmenopausal women on hormone replacement therapy and 38 postmenopausal controls, was performed. Plasma levels of prothrombin fragment 1 + 2 and thrombin-antithrombin III complex were measured by enzyme-linked immunosorbent assay. Multivariate analysis of the covariance was used for statistical analysis, controlling for patient's age because the hormone replacement therapy group was older. RESULTS: There were no statistically significant differences between the hormone replacement therapy and control groups in either of the clotting parameters measured. A comparison of the levels of prothrombin fragment 1 + 2 and thrombin-antithrombin III complex in patients receiving estrogen alone or estrogen plus progestin also revealed no differences. CONCLUSIONS: Current doses of postmenopausal hormone replacement therapy do not appear to enhance in vivo clotting. Thromboembolic complications among postmenopausal women receiving hormone replacement therapy may therefore be secondary to congenital or other acquired coagulation defects.

Initial arrest of tumor cells in the microvasculature and their attachment to the endothelium and subendothelial matrix (SEM) are essential prerequisites for metastasis to occur. Factors mediating these interactions are viewed as important determinants of the tumor-cell metastatic phenotype. In this work we have studied the effects of thrombin, its analogs and its precursors on the adhesive properties and metastatic potential of tumor cells. We show that alpha-thrombin, the native form of the key coagulation enzyme, is capable of enhancing tumor-cell adhesion to both the endothelium and SEM components represented by fibronectin. Subclotting, physiological concentrations of alpha-thrombin produced a 2- to 5-fold increase in tumor-cell adhesion. A bell-shaped dose-response curve was observed, with maximal effect at 0.1 U/ml. Maximum effect occurred when cells were exposed to the agonist for 15 min and exposure for up to 4 hr resulted in enhanced tumor-cell adhesion. Prolonged incubation with thrombin resulted in a decline in the thrombin-enhanced adhesion which reached unstimulated control levels by 24 hr. Thrombin precursors and active-site-inhibited thrombin analogs only had minimal adhesion-enhancing activity; nitro- and exosite-alpha-thrombin, which retain a functional active site, mimicked, although to a lesser degree, the action of alpha-thrombin. Tumor-cell incubation with thrombin resulted in an upregulated cell-surface expression of the alpha11b beta 3 integrin, a receptor mediating interactions between tumor cells and endothelial cells, and between tumor cells and SEM. Antibodies against alpha 11b beta 3 integrin effectively inhibited thrombin-enhanced tumor-cell adhesion. Thrombin effects on tumor cells involved the PKC signal transduction pathway as thrombin-enhanced adhesion was inhibited by pre-incubation with PKC inhibitors and a transient PKC translocation from cytosol to membrane was observed following thrombin challenge. In vivo, thrombin-treated tumor cells demonstrated a 2-fold increase in their lung-colonizing ability. In contrast to the adhesion results, the metastasis-enhancing effects of alpha-thrombin were mimicked by a thrombin precursor (prothrombin) and thrombin analogs.

Assays which detect the release of platelet-specific proteins and of peptides during thrombogenesis and are considered markers of activation of platelets and the coagulation system have recently been developed. This study was designed to utilize these haemostasis-related markers to test the hypothesis that a prethrombotic state is related to the presence, aetiology and severity of heart failure. Seventy patients with heart failure were evaluated and data were compared with 36 normal volunteers and 41 patients with coronary artery disease without heart failure (CAD). Thrombogenesis was documented using assays which measure platelet function, thrombin activity and fibrinolysis. Platelet function was measured by determining plasma concentrations of platelet factor 4 (PF4) and beta-thromboglobulin (BTG). Thrombin-antithrombin III complexes (TAT) and fibrinopeptide A (FPA) were determined to evaluate thrombin activity. Fibrinolytic activity was assessed by measuring D-Dimer levels. Patients with heart failure, when compared to normals, had increased plasma levels of BTG (89 +/- 62 IU.ml-1 vs 50 +/- 59 IU.ml-1, P < 0.01), TAT (4.6 +/- 4.3 micrograms.l-1 vs 2.3 +/- 0.64 micrograms.l-1, P < 0.005), and D-Dimer levels (506 +/- 444 IU.ml-1 vs 191 +/- 144 IU.ml-1, P < 0.0001). Patients with heart failure, when compared to the CAD group, had increased plasma levels of D-Dimer (506 +/- 444 ng.ml-1 vs 191 +/- 144 ng.ml-1, P < 0.05). Aetiology of heart failure did not affect these measurements. Patients with severe heart failure, as determined by high plasma norepinephrine concentration or low ejection fraction, were more likely to have activation of platelets and the coagulation system.(ABSTRACT TRUNCATED AT 250 WORDS)

The manual Heptest for measuring low-molecular-weight heparin fractions was applied to a fully automated, coagulation-dedicated analyzer, the Automated Coagulation Laboratory 300 Plus. The clot-based assay mode of the instrument was used, which operates on the principle of light scattering. Undiluted plasmas and the original reagents of the Heptest kit were used. Also, the 2-minute incubation time of the manual procedure was maintained. Automation reduced plasma and reagent volumes by about one half. As a result of the high precision of the automated procedure, single determinations suffice, and 18 plasma samples can be analyzed in about 8 minutes. Coefficients of variation were 1.0% to 3.2% for within-run and 1.9% to 6.0% for inter-run analyses. Analytical recovery was 98% to 104%. Comparisons of 132 samples between the two procedures yielded an R value of 0.974 for activity expression in seconds and 0.945 for U/mL. Several low-molecular-weight heparin fractions were tested.

Some traditional coagulation assays and several new molecular markers of hemostatic activation were measured in 37 patients with spinal cord injury (SCI). Twenty one of the patients (57%) developed deep vein thrombosis (DVT). The radiofibrinogen uptake test (RFUT) was used to diagnose DVT. Thirty eight percent of quadriplegic and 88% of paraplegic patients developed DVT (p < 0.005). No significant differences were found in platelet counts, mean platelet volumes, fibrinogen levels, von Willebrand factor (Ag) levels, platelet factor 4 and beta thromboglobulin concentrations between the groups with and without DVT. Fibrinopeptide A, thrombin/antithrombin III (TAT) complexes and plasma D-dimer levels were significantly higher in the patients with thrombosis. Most patients with DVT had elevated TAT complex levels up to three days before the RFUT became positive. D-dimer levels were highest after the diagnosis had been made.

The liver, the major site of production of most coagulation components, is intimately involved in the clearance of activation products. Hemostasic abnormalities relate to the extent of liver function impairment. Minor impairment may be associated with slight decreases in clotting factors, especially the vitamin Independent factors; major defects (cirrhosis) may display decreases of virtually all factors, impaired platelet function, and increased consumption (i.e., disseminated intravascular coagulation [DIC]). Liver surgery invariably leads to bleeding, most of which is caused by DIC.

The effect of laser irradiation on the thrombogenicity of thrombus was evaluated by treating thrombi, formed in-vitro from canine blood, with two different doses of cw Nd:YAG laser energy at 1064 nm. The thrombi were then incubated with whole blood, and the plasma levels of fibrinogen and thrombin-antithrombin III-complexes were measured. A statistically significant decrease (p0.05) in the thrombogenicity was indicated by a reduction in both fibrinogen consumption and levels of thrombin-antithrombin III-complexes in the high dose group (600 joules, 100 degrees C peak temperature) in comparison to the low dose group (300 joules, 70 degrees C peak temperature) and the untreated thrombi. These findings suggest that laser irradiation of thrombus at an appropriate dose may substantially reduce its thrombogenicity and ability to modulate hemostasis.

Chromogenic anti-Xa activity procedures were developed for monitoring LMW heparins on the Automated Coagulation Laboratory 300 Plus (ACL, Instrumentation Laboratory) system. For daily monitoring, a "Routine" procedure was devised which allows accurate measurements between plasma levels of 0.1 and 1.0 u/ml LMW heparin. For lower levels a "Routine-Low" method was developed which assesses activities between 0.05 and 0.4 u/ml. Due to variabilities in dODs of individual baseline plasmas, levels below 0.05 u/ml might be inaccurate when pooled normal plasma is used to establish the reference curve. While levels less than 0.05 u/ml should rarely be encountered when monitoring LMW heparins for routine clinical use, pharmacokinetic studies require accurate measurements below that level. For this reason a "Research-High" and a "Research-Low" procedure was designed. For these procedures a study subject's own baseline plasma was used to establish the reference curve. The "Research-High" measures activities between 0.4 and 2.0 u/ml, the "Research-Low" between zero and 0.4 u/ml. The procedures have excellent within-run and inter-run coefficients of variation (less than 5%) and high levels of accuracies. Even inter-instrumental reproducibilities are less than 10%. Different manufacturers' LMW heparins can be analyzed by these assays. The procedures offer full automation, great cost-effectiveness due to lower reagent volumes, rapid turn-around time and great accuracy and reproducibility.

Platelet factor 4 (PF4) and beta-thromboglobulin (BTG) are unique markers of irreversible platelet activation. We measured PF4 and BTG in amniotic fluid from 102 genetic amniocenteses, in which 78 had normal amniotic fluid alpha-fetoprotein (AFP) levels with normal pregnancies, and 24 had high amniotic fluid AFP levels with abnormal pregnancies. PF4 and BTG were significantly higher in the abnormal pregnancy/elevated amniotic fluid AFP group (p0.002 in each case) and correlated with AFP expressed as multiples of the median (p0.05 and p0.0001, respectively). Our results are compatible with passage of PF4 and BTG across fetal membranes and/or enhanced fetal platelet activation in fetuses with structural anomalies and elevated AFP.

Heparin-induced thrombocytopenia (HIT) and HIT thrombosis syndrome (HITTS) are immune-mediated complications of clinical use of unfractionated heparin (UFH). The antibody/antigen complex is composed of heparin and platelet factor 4. This complex not only activates platelets but also the clotting system leading to thrombin generation. This explains the thrombosing tendency of these patients, and venous and arterial thromboembolisms are encountered with a morbidity and mortality of about 25–37%. The incidence of HIT is about 3% when UFH is administered therapeutically. The diagnosis is at this time based on clinical observations, especially a sudden, unexplained drop in platelet counts without other reasons. Laboratory tests can be used to confirm the clinical diagnosis, but none of the available tests is 100% reliable. There is no test that will predict HIT and no test that will signal the development of HITTS. Treatment consists of discontinuation of UFH in any form and anticoagulation with danaparoid or r-hirudin, if needed. The use of low molecular weight heparins instead of UFH could largely (not totally) alleviate the problem.

Objective: This study was undertaken to evaluate the effects on hemostatic factors of a low-dose preparation of levonorgestrel and ethinyl estradiol in a 12-cycle study. Study Design: Thirty healthy women began taking 100 μg levonorgestrel and 20 μg ethinyl estradiol on the first day of the menstrual cycle, continued to take the preparation for the next 21 days, and then took placebo for 7 days. Mean changes in prothrombin time, partial thromboplastin time, and levels of factors VII and X, antithrombin, plasminogen, fibrinogen, protein S, thrombin-antithrombin complexes, and D-dimer were analyzed at baseline and at cycles 3, 6, and 12 with paired Student t tests. Results: Factor X, plasminogen antigen and activity, and D-dimer levels were significantly increased ( P ≤ .01) during all 3 cycle periods. Antithrombin antigen and protein S total antigen levels were significantly ( P ≤ .001 ) decreased at cycles 3, 6, and 12, whereas factor VII and protein S activity levels were significantly ( P ≤ .05) decreased at cycle 3 and at cycles 3 and 6, respectively. Conclusion: The effects on hemostatic factors in healthy women of a monophasic preparation of 100 μg levonorgestrel and 20 μg ethinyl estradiol were similar to those of other low-dose oral contraceptives. (Am J Obstet Gynecol 1999;181:S63-6.)

The sticky platelet syndrome (SPS) is an autosomal dominant platelet disorder associated with arterial and venous thromboembolic events. It is characterized by hyperaggregability of platelets in platelet-rich plasma with adenosine diphosphate (ADP) and epinephrine (type I), epinephrine alone (type II), or ADP alone (type III). Clinically, patients may present with angina pectoris, acute myocardial infarction (MI), transient cerebral ischemic attacks, stroke, retinal thrombosis, peripheral arterial thrombosis, and venous thrombosis, frequently recurrent under oral anticoagulant therapy. Clinical symptoms, especially arterial, often present following emotional stress. Combinations of SPS with other congenital thrombophilic defects have been described. Low-dose aspirin treatment (80 to 100 mg) ameliorates the clinical symptoms and normalizes hyperaggregability. The precise etiology of this defect is at present not known, but receptors on the platelet surface may be involved. Normal levels of platelet factor 4 (PF4) and beta-thromboglobulin in plasma suggest that the platelets are not activated at all times; they appear to become hyperactive upon ADP or adrenaline release. In vivo clumping could temporarily or permanently occlude a vessel, leading to the described clinical manifestations. The syndrome appears to be prominent especially in patients with unexplained arterial vascular occlusions.