Elsevier

Thrombosis Research

Volume 83, Issue 5, 1 September 1996, Pages 363-373
Thrombosis Research

APC-RESISTANCE AS MEASURED BY A TEXTARIN TIME ASSAY: COMPARISON TO THE APTT-BASED METHOD

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Abstract

Protein C is a major regulatory protein critical to physiologic anticoagulation. When activated, it selectively degrades the activated forms of factors V and VIII, thereby, down-regulating blood coagulation. Using an activated partial thromboplastin time (APTT) assay, Dahlback et al. recently reported that some individuals with thrombophilia show a poor in vitro anticoagulant response to activated protein C (APC-Resistance). Subsequent studies identified a point mutation in the gene for factor V as the underlying cause of APC-Resistance. The incidence of APC-Resistance in patients with recurrent thromboembolic events approaches 50%. The APC-Resistance phenotype is also present in approximately 5% of normal Caucasian subjects. In an attempt to develop a more sensitive and specific test system, we evaluated an assay based on Textarin®(Pentapharm, Basel, Switzerland). Textarin®, a protein fraction of Pseudonaja textilis venom (Australian Eastern Brown Snake) activates prothrombin in the presence of phospholipid (PL), factor V and calcium ions. Based on Textarin's requirement for factor V, we developed a Textarin time assay to test for APC-Resistance. We evaluated this test system in normal subjects and the following patient populations: stable orally anticoagulated, previously diagnosed factor V Leiden, and therapeutically heparinized samples. We found the Textarin assay to be a sensitive and specific test system to identify APC-Resistance. The phenotypic Textarin APC-Resistance test correlated more closely with the genotypic abnormality of factor VR506Q than the APTT-APC-Resistance test.

Section snippets

Blood Collection, Preparation and Storage

Blood was collected in evacuated tubes (Vacutainer Systems®, Becton Dickinson, Rutherford, New Jersey) containing 0.5 ml of 0.105M buffered sodium citrate. Platelet poor plasma (PPP) was prepared by centrifugation at 2000 g for 10 min. in a 4°C centrifuge. The PPP supernatant was then transferred to a 5 ml polypropylene tube using a plastic transfer pipette and recentrifuged under the same conditions. The final PPP product was transferred to another 5 ml polypropylene tube and tested within 4

Oral Anticoagulated Samples

After determining the reference intervals as described in the previous section, seventeen stable oral anticoagulated samples were tested in both test systems for comparison. The patient INRs ranged from 1.3 to 4.0. Of these seventeen patients, two tested positive for APC resistance (ratio <2.0) in both test systems. (Table 1) These patients were then confirmed to be heterozygous factor V Leiden using Polymerase Chain Reaction (PCR) [11].

Previously Diagnosed Factor V Leiden Samples

Fourteen samples from patients who had been previously

DISCUSSION

Since the discovery of APC-Resistance as a major cause of venous thrombosis, laboratories have routinely implemented tests to identify these patients. The most popular test system employs the original assay as described by Dahlback and colleagues utilizing an APTT system [1]. However, other test systems have been utilized including: a dilute Russell Viper Venom Time, a modified prothrombin time (one-stage tissue factor-dependent factor V assay), a protein C activation dependent clotting time

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