Randall S. Miller

Factors Contributing to Individual Propensity for Arterial Thrombosis

Objective—Occurrence of arterial thrombosis secondary to vascular disease in an individual is not easily predicted. After establishing that this poor predictability arises at least in part from an intrinsic thrombosis propensity of the individual, we sought to determine whether the propensity for arterial thrombosis is governed by blood or arterial wall factors. Methods and Results—To evaluate the variability arising from the blood, autologous 111In-labeled platelet deposition was measured after high-shear perfusion of compressed aortic strips, prepared from a single pig, with heparinized blood from 25 pigs. To evaluate the variability arising from the vessel wall, aortic strips from 8 pigs were superfused with blood from a single animal. Blood samples from 25 animals superfused over aortic substrate from a single source yielded a 24-fold range of platelet deposition. In contrast, when aortic substrates from 8 different animals were superfused with blood from a single animal, platelet deposition spanned a 3-fold range. Platelet deposition was significantly correlated with whole-blood lymphocyte counts and with platelet counts . Conclusions—Individual propensity for arterial thrombosis in pigs is more greatly influenced by blood components than by elements within the arterial wall.

Precision and Reliability of 5 Platelet Function Tests in Healthy Volunteers and Donors on Daily Antiplatelet Agent Therapy

BACKGROUND Anticoagulation protocols used during mechanical circulatory support call for titration of antiplatelet agents. We compared the precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet therapy to distinguish their efficacy for titrating antiplatelet therapy. METHODS We assessed arachidonic acid-induced platelet function by light transmission aggregometry (LTA), Multiplate impedance aggregometry, VerifyNow, and platelet mapping by thromboelastography (TEG PM). We assessed ADP-induced platelet function by the same methods and flow cytometry. Forty healthy volunteers and 10-13 volunteers on daily aspirin and/or clopidogrel therapy were evaluated. We compared tests for intraassay precision, interassay precision (samples from 2 separate blood draws), and reliability coefficient. RESULTS For arachidonic acid-induced platelet aggregation in healthy volunteers, intra- and interassay CVs were ≤ 10% for all methods. Intra- and interassay precision among donors on daily aspirin was ≤ 30% for all methods except LTA (38% interassay CV) and TEG PM (95% intraassay and 104% interassay CV). For ADP-induced platelet function, intra- and interassay precision was ≤ 10% and ≤ 30% for all methods. Only Multiplate demonstrated moderate or greater (R > 0.40) reliability coefficients for arachidonic acid-induced platelet function among all subjects. All methods of ADP-induced platelet function, except TEG PM, demonstrated substantial or greater (R > 0.60) reliability among all subjects. CONCLUSIONS TEG PM is least suited to monitor effects of antiplatelet agents. Multiplate impedance aggregometry was the only method to demonstrate an acceptable reliability coefficient among healthy volunteers and donors on both aspirin and clopidogrel therapy.

Tissue Prothrombin Universal Distribution in Smooth Muscle

Immunohistochemical analysis of surgically obtained porcine tissue samples reveals ubiquitous staining for prothrombin in organs rich in smooth muscle content and universal staining of smooth muscle in tissue vasculature. The native character of tissue prothrombin is verified first by chromogenic substrate hydrolysis and hirudin inhibition after incubation of tissue extracts with taipan snake venom and phospholipid. Western analysis of tissue extracts confirms the native zymogen molecular weight. In addition, prothrombin purified in good yield from porcine uterus is activated by Echis carinatus venom which, like taipan venom, is 4-carboxyglutamic acid-sensitive. After correction for blood (gross heme) and interstitial fluid (albumin), excess functional prothrombin is observed in extracts of tissues having abundant smooth muscle. In contrast with factor X, the yield of prothrombin purified from porcine uterus greatly exceeds that attributable to contamination by whole blood. Northern blot analysis from selected bovine tissues extracted for polyadenylated messenger RNA is equivocal for prothrombin mRNA with the exception of liver, which is positive. It is concluded that functionally intact prothrombin is widely distributed among tissues owing to smooth muscle content, although the mechanism of emplacement and physiologic significance of prothrombin in these tissues remains unclear.

Monoclonal antibodies selective for the functional states of bovine factor V and factor Va

Hybridoma technology has been used for the production of murine monoclonal antibodies to bovine coagulation Factor V and its thrombin-activated product, Factor Va. Hybrid cell cultures were assayed for the production of anti-Factor V and anti-Factor Va antibodies by a solid-phase radioimmunoassay. Antibody-producing cell lines were selected, cloned and grown as ascites tumors. Gel filtration chromatography (Ultrogel AcA34) and affinity chromatography (protein A-Sepharose) were used to isolate the monoclonal immunoglobulins from the ascites fluids. Thirteen monoclonal antibodies have been characterized with respect to their binding to Factor V and Factor Va and their effect on cofactor bioactivity. Six of these thirteen antibodies react with both Factor V and Factor Va. One of these antibodies is strongly inhibitory, while a second antibody is only moderately inhibitory. The antibody produced by another cell line binds Factor V but not Factor Va and is not inhibitory. The remaining six cell lines each produce an antibody that reacts preferentially with Factor Va, and each of these antibodies is inhibitory to some extent. Both a radioimmunoassay and light scattering have been used to study the interaction of the immunoglobulins with Factor V and Factor Va. The light scattering technique has proven useful to study the interaction of isolated antibodies and antigens and permits the determination of interaction stoichiometries. Each of the interactions studied was characterized by a stoichiometry of two antigens per antibody. These monospecific immunochemical reagents will be useful in the study of structure and function relationships of Factor V, Factor Va and activation fragments.

Inhibition and reversal of platelet‐rich arterial thrombus in vivo: direct vs. indirect factor Xa inhibition

Summary.  Background/objective: The efficacy of a direct factor (F)Xa inhibitor, ZK‐807834, was compared with indirect inhibition by enoxaparin for inhibition and deaggregation of acute platelet‐rich thrombi in a well‐characterized porcine carotid injury model. Methods: A crush injury was performed on a randomly chosen carotid artery and the thrombus allowed to propagate for 30 min. Pigs then received intravenous drug for 35 min: ZK‐807834‐Dose 1 (40 µg kg−1 bolus + 1.5 µg kg−1 min−1 infusion, n = 6); ZK‐807834‐Dose 2 (20 µg kg−1 bolus + 0.75 µg kg−1 min−1 infusion; n = 6); enoxaparin (1 mg kg−1 bolus; n = 6); or saline (n = 6). Five minutes after drug initiation, the contralateral artery was injured. Thrombus size was monitored by scintillation detection of autologous 111In‐platelets. Results: The prothrombin time ratio was 2.2 ± 0.1; 1.4 ± 0.3; 1.2 ± 0.9 and 1.1 ± 0.2, respectively. ZK‐807834‐Dose 1 significantly inhibited carotid platelet deposition (525 ± 226 × 106 cm−2; P = 0.008), whereas ZK‐807834‐Dose 2 (2325 ± 768) and enoxaparin (1236 ± 383) were not different from saline (2776 ± 642). Thrombus deaggregation was greatest for animals receiving ZK‐807834‐Dose 1 (473 ± 185). Neither ZK‐807834‐Dose 2 (1588 ± 480) nor enoxaparin (1618 ± 686) was different from saline control (2222 ± 598). Conclusions: Direct FXa inhibition with ZK‐807834, at a prothrombin time ratio of 2.2, effectively inhibits thrombosis and promptly deaggregates thrombi induced by arterial injury. In contrast, indirect FXa inhibition with enoxaparin was ineffective.