Richard A. Marlar

Diagnosis and treatment of homozygous protein C deficiency. Report of the Working Party on Homozygous Protein C Deficiency of the Subcommittee on Protein C and Protein S, International Committee on Thrombosis and Haemostasis.

This report summarizes the documented cases of homozygous protein C deficiency in the United States and Europe. Procedures for diagnosing and treating this disorder (both initially and over the long term) have been compiled by a working party on homozygous protein C deficiency of the Subcommittee on Protein C of the International Committee on Thrombosis and Haemostasis. Homozygous protein C deficiency is an autosomal recessive disorder that usually manifests itself by purpura fulminans and, less commonly, by massive large vein thrombosis; severe diffuse intravascular coagulation also develops in these infants, and there is evidence of intrauterine thrombosis. For confirmation of homozygous protein C deficiency in a neonate with purpura fulminans or massive venous thrombosis, the infant should have undetectable protein C activity and both parents should be heterozygous for protein C deficiency. At the onset of symptoms, the initial treatment should be plasma (8 to 12 ml/kg every 12 hours) until all lesions have healed. Two modalities for long-term treatment are accepted as useful in these children: oral anticoagulant therapy or protein C replacement (fresh frozen plasma or prothrombin complex concentrate). Liver transplantation has been performed in only one child, with success. Oral anticoagulation (vitamin K antagonists, maintaining the prothrombin time from one and one-half to two times control values or at the international Normalized Ratio of 2.5 to 4.4) is our recommendation of choice for long-term treatment. With appropriate care, these children are able to be free of coagulopathy and live relatively normal lives.

Severe homozygous protein C deficiency

An infant with recurrent purpura fulminans in the first year of life was found to have severe homozygous deficiency of protein C (less than 1% of normal levels). The episodes of purpura fulminans were controlled by infusions of fresh frozen plasma containing protein C. The requirement of frequent plasma infusions, however, eventually resulted in several complications secondary to hyperproteinemia. Factor IX concentrates rich in protein C were then given to maintain adequate levels of the factor while minimizing the amount of extraneous proteins. The patient has remained asymptomatic and free of complications for greater than 10 months while receiving these concentrates every 48 hours.

High-affinity RNA ligands to human α-thrombin

Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to isolate from a population of 10(13) RNA molecules two classes of high affinity RNAs that bind specifically to human alpha-thrombin. Class I RNAs are represented by a 24-nucleotide RNA (RNA 16.24), and class II RNAs are represented by a 33-nucleotide RNA (RNA 27.33). RNA 16.24 inhibits thrombin-catalyzed fibrin clot formation in vitro. Secondary structures are proposed for these RNAs, revealing a novel stem-loop structure for RNA 16.24, comprised of an unusually large 16-nucleotide loop. Mutants of RNA 16.24 were generated to investigate structural features critical to high-affinity binding. Phosphate modification with ethylnitrosourea identified regions of the RNAs necessary for electrostatic interactions. Competition with heparin suggests that these RNAs bind the electropositive heparin-binding site of thrombin. These ligands represent a novel class of thrombin inhibitors that may be suitable for therapeutic or diagnostic applications.

Severe neonatal protein C deficiency: Prevalence and thrombotic risk

Severe deficiencies of protein C, a pivotal coagulation-regulatory protein, have been reported in neonates as an apparently transient condition. In this prospective study, cord blood was collected at 193 deliveries and assays of protein C were correlated with clinical status, other coagulation results, and outcome. Protein C levels of less than 0.1 unit/ml were found most frequently in preterm infants with respiratory distress, infants of diabetic mothers, and infants of twin gestations. Levels of protein C correlated with levels of factor VIII activity but did not correlate with markers of consumptive coagulopathy. A protein C level less than 0.1 unit/ml was significantly correlated with the subsequent onset of thrombosis, even when the effects of gestational age and birth weight were excluded. Low cord blood levels of protein C may reflect delayed maturation or increased turnover in certain infants and appear to convey an independent risk of thrombosis, but the critical concentration of protein C necessary to maintain neonatal hemostasis is not known.

The effect of time and temperature variables on routine coagulation tests.

This study evaluates the effects of time and temperature variables on routine coagulation assays [Prothrombin Time test and Activated Partial Thromboplastin Time (APTT) test]. Four different groups were studied: healthy volunteers, hospitalized patients not receiving anticoagulants, patients receiving oral anticoagulant therapy and patients receiving unfractionated heparin therapy. Samples were subjected to one of four conditions: (1) centrifuged immediately and stored at room temperature (20–22°C); (2) centrifuged immediately and stored on ice (4°C); (3) stored as whole blood without centrifugation, at room temperature and (4) stored without centrifugation, on ice. Coagulation tests were performed as soon as possible after phlebotomy and at specified times up to 24 h. Our data demonstrate that prothrombin time results are stable for up to 24 h, remaining constant regardless of storage conditions. APTT assays are stable for up to 8 h, except for patients receiving unfractionated heparin therapy. Heparinized samples, when stored uncentrifuged at room temperature, demonstrate a clinically significant shortening of the APTT and individual samples demonstrate a greater than 50% decrease in ex-vivo heparin levels at 4 h.

Heparin levels to guide thromboembolism prophylaxis during pregnancy

OBJECTIVE Our purpose was to determine the dose of heparin required in pregnant women to achieve the same heparin levels as standard doses of 5000 units given subcutaneously every 12 hours in the nonpregnant population. STUDY DESIGN Fourteen pregnant women placed on heparin prophylaxis for a history of thromboembolism had blood drawn for 64 anti-Xa level determinations in the second and third trimesters. Heparin doses were adjusted in an attempt to achieve a midinterval or peak level of 0.05 to 0.25 U/ml, which corresponds to the range seen in nonpregnant patients given standard doses of 5000 units subcutaneously every 12 hours. RESULTS A standard heparin dose of 5000 units given subcutaneously every 12 hours was inadequate to achieve the desired range in this pregnant population. In five of nine second-trimester pregnancies 7500 units given subcutaneously every 12 hours was inadequate to attain this range. In six of 13 third-trimester pregnancies, > 10,000 units subcutaneously every 12 hours was needed. CONCLUSIONS Heparin requirements may increase and are highly variable in patients during pregnancy. Until appropriate clinical outcomes trials can determine optimal dosing, measuring anti-Xa activity may be useful to guide therapy.

Protein C and protein S levels in two patients with acquired purpura fulminans

Purpura fulminans (PF) is a cutaneous manifestation of a dramatic and deadly syndrome of systemic disseminated intravascular coagulation (DIC). It is characterized by microvascular thrombosis in the dermis followed by perivascular haemorrhage. Since two other related syndromes involve the protein C (PC) system, we undertook a serial study to investigate the levels of PC and protein S (PS) in two patients with acquired PF. Laboratory findings were consistent with DIC, and both patients were treated with blood replacement and heparin therapy. The levels of PC activity were very low during the initial 24–36 h after onset and gradually increased until returning to normal levels. The total and ‘free’PS were also abnormal during the initial onset of PF. The total and free PS increased to normal after 4–6 d. Although the pathogenesis is not fully understood, the infection and sepsis appears to consume PC and PS selectively during the PF and DIC phase. Acquired PF appears to selectively involve the PC system in a similar fashion to two other syndromes of PF‐like lesions.

Activated Protein C Resistance Assay Detects Thrombotic Risk Factors Other Than Factor V Leiden

Activated protein C (APC) resistance is a common risk factor for venous thromboembolism (VTE) attributed to various mechanisms, including factor V Leiden (FVL) polymorphism. FVL is considered responsible for up to 95% of APC resistance; however, other factor V polymorphisms and elevated factor VIII levels also have been implicated. We assessed whether additional mechanisms contribute to APC resistance in a blinded case-control study of 65 subjects by measuring APC resistance using 3 methods: 2 activated partial thromboplastin time-based methods with and without dilution in factor V-deficient plasma and 1 Russell viper venom-based assay (RVV). Without factor V-deficient plasma, 24 subjects were APC resistant; with factor V-deficient plasma, the assay identified fewer APC-resistant subjects, as did RVV. All assays detected the 7 heterozygous FVL subjects. Thirteen subjects had factor VIII levels above 150% (1.50). After excluding subjects with FVL or elevated factor VIII levels, 4 subjects still had APC resistance. VTE risk trended higher for subjects with APC resistance in the absence of FVL. Measurement of APC resistance without dilution in factor V-deficient plasma is needed to assess for potentially important thrombotic risk factors other than FVL.

Developmental expression of protein C and protein S in the rat

In order to better understand the expression of the Protein C/Protein S anticoagulant system, we have isolated and characterized cDNAs coding for rat Protein C and Protein S. These cDNAs were used in Northern analysis to determine tissue-specificity and developmental expression patterns for mRNAs coding for Proteins C and S. In rats, Protein C mRNA is expressed almost exclusively in liver with a small amount of expression in kidney, diaphragm, stomach, intestine, uterus and placenta. Protein C mRNA was not expressed in brain, heart, lung, spleen, small intestine, large intestine, ovary, or urinary bladder. In liver, Protein C mRNA is expressed at very low levels at prenatal day 18 and these levels increased to maximal levels by postnatal day 13. The size of the mRNA coding for rat Protein C is approximately 1.9 kb. Rat Protein S mRNA was expressed in all tissues examined: brain, heart, lung, diaphragm, liver, spleen, stomach, small intestine, large intestine, kidney, adrenal ovary, uterus, placenta, and urinary bladder. Interestingly, there were 4 bands hybridizing with the rat protein S cDNA that were evident in many of the tissues examined, corresponding to mRNA sizes of approximately 3.5, 2.6, 1.8, and 0.3 kb. There was a difference in tissue-specificity of each mRNA. The 1.8 kb band is generally the most prominent autoradiographic band in any tissue. From these results, it is evident that the expression of Protein C mRNA is similar to that of other vitamin K-dependent proteins. The expression of Protein S mRNA, however, is surprisingly complex and may include alternative splicing of mRNA to generate the various sizes evident on Northern analysis.

Recombinant human protein C: Comparative functional studies with human plasma protein C

Protein C (PC) is the central protein in a major antithrombotic regulatory mechanism. Hereditary deficiencies of PC are associated with thrombosis. Therapeutic PC replacement may be an important treatment if pure functional human protein C is available in sufficient quantity. Human PC has been produced on a commercial scale using recombinant techniques. To study the functional properties of recombinant protein C (r-PC), we undertook a comparative investigation of the basic properties of r-PC and plasma protein C (n-PC). Both were isolated by immunopurification methods. Protac C activation proceeded at the same rate and kinetics for both forms. With thrombin-thrombomodulin (T-TM) activation, r-PC is significantly better than the activation of n-PC (for r-PC: Kcat/Km = 378 vs. n-PC: Kcat/Km = 35). No difference in the anticoagulant (aPTT prolongation) or profibrinolytic activities (inactivation of PAI-1 and PAI-3) were observed between activated r-PC and n-PC. Based on these functional studies, recombinant protein C has similar properties to the plasma form of protein C. However, T-TM activation of r-PC occurs faster than the n-PC. The mechanism is unknown, but may be due to the presence of larger amounts of single chain protein C which exists in a conformation more rapidly activated by the T-TM complex.