Quantitative immunoblotting assay of blood coagulation factor XII

The immunoblotting technique was applied to the study of Factor XII (F.XII) in plasma. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma followed by electroblotting of the electropherograms to nitrocellulose (NC) membranes and immunologic detection by a double antibody technique was used. ¹²⁵-F.XII was transferred to the NC membrane in amounts proportional to the amount applied to the gel provided that a constant amount of carrier protein was present. Based on this, a quantitative assay was developed using either normal plasma or F.XII dilutions in F.XII-deficient plasma as standards. The measurement of F.XII antigen by immunoblotting was reproducible and gave values similar to those obtained by radial immunodiffusion. Two normal plasma pools contained 26 and 29 μ/ml of F.XII according to the immunoblotting assay. Compared to other immunoassays, immunoblotting has the advantage of directly estimating the apparent molecular weight (MW) of the protein of interest. Thus, we could confirm the normal apparent MW (80,000) of a F.XII-like molecule previously isolated from a cross reacting material (CRM)-positive F.XII-deficient plasma. None of eight CRM-negative F.XII-deficient plasmas showed an 80,000 MW immunoreactive molecule. However, five of these eight plasmas had a faint autoradiographic band at 115,000 MW that was similarly seen in only three out of 43 individual normal plasmas. The nature of this 115,000 MW band remains to be defined.     

A quantitative dot immunobinding assay for coagulation factor XII in plasma

A dot immunobinding assay on nitrocellulose (NC) membranes has been developed for the quantification of human coagulation factor XII (F XII). Plasma samples were dotted on to NC filters and F XII was detected using a polyclonal antiserum followed by a radiolabelled antigen overlay. Dilutions of either pooled normal human plasma (NHP) or purified F XII in F XII deficient plasma were used as standards. Quantification was performed by measuring the radioactivity of bound 125I-F XII. Precise measurements of F XII antigen (F XII: Ag) were possible with a sensitivity down to 0.12 ng. Thus, dotting samples containing 0.5 microliter of plasma permitted detection of a F XII concentration corresponding to 1% of the level in NHP. The intra-assay coefficient of variation (CV) was less than 5% and the interassay CV was less than 16%. F XII:Ag in plasma samples of 50 healthy adults ranged from 12 micrograms/ml to 47 micrograms/ml. A good correlation (r = 0.93) existed between F XII:Ag and F XII clot promoting activity (F XII:C) in these samples. NHP contained 24.1 micrograms/ml F XII:Ag confirming earlier results obtained by other methods. In 16 pregnant women levels of F XII:Ag as well as of F XII:C were elevated, but F XII:Ag was disproportionately higher compared with F XII:C. The immunobinding assay has the following advantages: (1) rapid quantification of large numbers of samples is possible, (2) the sensitivity down to 1% of NHP is better than that of several other methods, (3) only very small amounts of both test material and reagents are needed.     

Congenital thrombotic thrombocytopenic purpura caused by new compound heterozygous mutations of the ADAMTS13 gene

Upshaw–Schulman syndrome (USS) is due to severe congenital deficiency of von Willebrand factor (VWF)‐cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 domains, nr 13) activity resulting in the presence of unusually large forms of VWF in the circulation, causing intravascular platelet clumping and thrombotic microangiopathy. Our patient, a 26‐year‐old man, had attacks of thrombotic thrombocytopenic purpura (TTP) with thrombocytopenia and a urine dipstick positive for hemoglobin (4+), often as the only sign of hemolytic activity. He had ADAMTS13 activity of <1% of normal plasma without the presence of inhibitors of ADAMTS13. ADAMTS13 deficiency was caused by two new mutations of the ADAMTS13 gene: a deletion of a single nucleotide in exon17 (c. 2042 delA) leading to a frameshift (K681C fs X16), and a missense mutation in exon 25 (c.3368G>A) leading to p.R1123H. This case report confirms the importance of the analysis of the ADAMTS13 activity and its inhibitor in patients who have episodes of TTP, with a very low platelet count and sometimes without the classic biochemical signs of hemolysis.     

Plasma therapy in thrombotic thrombocytopenic purpura: review of the literature and the Bern experience in a subgroup of patients with severe acquired ADAMTS-13 deficiency

Based on clinical studies daily plasma exchange (PE) has become the first-choice therapy for thrombotic thrombocytopenic purpura (TTP) since 1991. Recent findings may explain its effectiveness, which particularly may include supply of ADAMTS-13 and removal of anti-ADAMTS-13 autoantibodies and unusually large von Willebrand factor (VWF) multimers. The most preferable PE regimens as well as replacement fluids are discussed and treatment-related adverse reactions are summarized. Proposals for a potential reduction of their frequency and for improvement of treatment efficiency are given. These suggestions are partially based on the experience of our institution in adult patients with severe ADAMTS-13 deficiency (<5% activity), and include (1) continuous calcium-gluconate infusion during PE in order to reduce citrate-related adverse reactions; (2) the evaluation of solvent/detergent-treated (S/D) plasma as replacement fluid in order to reduce adverse events due to fresh frozen plasma (FFP); (3) the evaluation of immunoadsorption in order to increase procedural efficiency in autoantibody removal; and (4) the substitution of ADAMTS-13 by means of recombinant drug instead of plasma.     

von Willebrand factor-cleaving protease (ADAMTS-13) activity determination in the diagnosis of thrombotic microangiopathies: the Swiss experience

Severe deficiency of von Willebrand factor (VWF)-cleaving protease (ADAMTS-13) activity (<5% of normal) is a specific finding for acute idiopathic thrombotic thrombocytopenic purpura (TTP), a disorder that presents as thrombocytopenia, microangiopathic hemolytic anemia, and often organ dysfunction such as neurological disturbances or renal failure, and fever. Between January 2001 and July 2003, ADAMTS-13 activity was determined in plasma samples of 396 consecutive patients referred to our laboratory for diagnostic purposes. Plasma samples with ADAMTS-13 activity less than 5% were in addition tested for the presence of inhibitory antibodies. Patients were assigned to 10 predefined clinical categories according to information provided by the referring clinician: thrombotic microangiopathy (TMA) not further specified; neoplasia- or chemotherapy-associated TMA; TMA following hematopoietic stem cell transplantation; TMA with additional/alternative disorder; idiopathic TTP; hemolytic-uremic syndrome (HUS) not specified; HUS with diarrhea prodrome (D+HUS); atypical HUS; other hematological disorder; and no clinical information available. Severe ADAMTS-13 deficiency was found in 69 (17%) patients, including 42 with acquired idiopathic TTP, either at initial presentation or at relapse, 14 with confirmed or suspected hereditary TTP, 10 with TMA not further specified, two with neoplasia- or chemotherapy-associated TMA, and one in continued clinical remission 3.4 years after splenectomy for plasma-refractory TTP. Forty-three (62%) patients with ADAMTS-13 activity less than 5% displayed inhibitory antibodies. Severe ADAMTS-13 deficiency was found in 60% of patients diagnosed with acute idiopathic TTP, but in none of 130 patients diagnosed with HUS or in any of the 14 patients with hematopoietic stem cell transplantation-associated TMA. Thus, plasma ADAMTS-13 activity less than 5% does not identify all patients clinically diagnosed with TTP, and severe ADAMTS-13 deficiency is not invariably associated with clinical manifestations of microvascular platelet clumping.     

Ten years of prophylactic treatment with fresh-frozen plasma in a child with chronic relapsing thrombotic thrombocytopenic purpura as a result of a congenital deficiency of von Willebrand factor-cleaving protease

We report the results of 10 years of prophylactic fresh-frozen plasma (FFP) infusion therapy in a 14-year-old girl with chronic relapsing thrombotic thrombocytopenic purpura (TTP), in whom a severe congenital von Willebrand factor (VWF)-cleaving protease deficiency has been documented. Severe haemolytic crises triggered by infections were prevented and her present renal and neurological functions have been preserved. Sequential measurements of protease activity and platelet count after FFP infusion led us to conclude tentatively that 5% may be sufficient to degrade very large and adhesive VWF multimers.     

Methodology and clinical significance of heparin cofactor II. Probable heparin cofactor II deficiency in a patient with cerebrovascular thrombosis

HC II was functionally determined by thrombin inhibition in the presence of heparin in AT III-free plasma prepared by immunoadsorption on anti-AT III-Sepharose 4B column. HC II antigen concentration was assayed using specific antibodies to HC II. Simultaneously, AT III was measured. Plasma levels of HC II and AT III were determined in 110 patients with thrombotic tendency and two patients with obstetric complications and DIC. Highly significant correlations between activity and antigen prove the suitability of the methods. Reduced levels of HC II to about 50% with normal AT III values were repeatedly found in one patient with thrombotic tendency. The course of AT III and HC II during the process of DIC suggests that HC II may function as a thrombin inhibitor reserve when AT III becomes subnormally low.     

Triplet structure of von Willebrand factor reflects proteolytic degradation of high molecular weight multimers.

High molecular weight (HMW) and low molecular weight (LMW) forms of von Willebrand factor (vWF) were isolated from normal human plasma in the presence of protease inhibitors. HMW and LMW vWF preparations were subjected to reduction of interdimeric disulfide bridges under mild reducing conditions. Following sodium dodecyl sulfate electrophoresis in 3% agarose, the vWF bands were detected by immunoblotting with a polyclonal rabbit anti-vWF antiserum as well as with two monoclonal antibodies directed against epitopes located in the NH2-terminal (MAb 418) or in the COOH-terminal (MAb 9) region of the vWF subunit. Our results suggest that the slowest migrating band of the dimeric triplet set of LMW vWF represents an asymmetric structure composed of an intact subunit to which one NH2-terminal and one COOH-terminal fragment are linked by disulfide bridges. The intermediate band of the first triplet of LMW vWF strongly reacted with MAb 9 but not with MAb 418, indicating that it represents a dimer of COOH-terminal fragments. The fastest migrating band of the same triplet is apparently a dimer of the NH2-terminal fragments because it reacted with MAb 418 but not with MAb 9. Each next higher family of triplets seems to contain one more asymmetric fragment of dimeric size. These results are compatible with a model according to which LMW forms of vWF are derived from HMW vWF by proteolytic cleavage in the circulating blood.     

Circulating DNA and myeloperoxidase indicate disease activity in patients with thrombotic microangiopathies

Thrombotic microangiopathies (TMAs) are a group of life-threatening disorders characterized by thrombocytopenia, fragmentation of erythrocytes, and ischemic organ damage. Genetic disorders, autoimmune disease, and cancer are risk factors for TMAs, but an additional, unknown trigger is needed to bring about acute disease. Recent studies suggest that DNA and histones are released during inflammation or infection and stimulate coagulation, thrombosis, thrombocytopenia, and organ damage in mice. We show that extracellular DNA and histones as well as markers of neutrophils are present in acute TMAs. Analysis of plasma from TMA patients of different clinical categories revealed elevated levels of DNA-histone complexes and myeloperoxidase (MPO) from neutrophil granules as well as S100A8/A9, a heterocomplex abundant in neutrophil cytosol. During therapy of thrombotic thrombocytopenic purpura, a subtype of TMAs often associated with severe ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) deficiency, plasma DNA and MPO were inversely correlated with platelet counts, and their levels indicated amelioration or exacerbation of the disease. ADAMTS13 deficiency together with increased levels of plasma DNA and MPO were characteristic for acute thrombotic thrombocytopenic purpura. A minor infection often precedes acute TMA and extracellular DNA and histones released during the inflammatory response could provide the second hit, which precipitates acute TMA in patients with pre-existing risk factors.