The genes for SHBG/ABP and the SHBG-like region of vitamin K-dependent protein S have evolved from a common ancestral gene.

Sex hormone binding globulin (SHBG) is the most important sex steroid transport protein in human plasma. It is the product of the same single gene as the androgen binding protein (ABP) of testis. Protein S is another protein, which is an important cofactor in the anticoagulation system and, as far as is known today, functionally unrelated to SHBG/ABP. Protein S also has a role in the complement system. A comparison of the human genes for SHBG/ABP and protein S reveals a sequence similarity, which is of a low grade only, between the SHBG/ABP protein and a similar sized COOH-terminal domain of protein S. However, the intron-exon organization exhibits a striking similarity in the two genes, illustrating evolutionary events leading to the appearance of two functionally different proteins from common ancestral genetic elements.

Adenine and guanine nucleotide metabolism during platelet storage at 22 degrees C.

Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C-adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation. All of these data should be helpful in improving the function and viability of PC as currently stored for 5 days, in devising methods for storage beyond 5 days, and in the development of synthetic media for PC storage.

Molecular analysis of the gene for vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization.

Protein S is a vitamin K dependent plasma protein and a cofactor to activated protein C, a serine protease that regulates blood coagulation. The haploid genome contains two protein S genes (alpha and beta) with the protein S alpha-gene corresponding to the cloned cDNA. We have now isolated and mapped overlapping genomic clones that cover an area of 50 kilobases of the protein S alpha-gene which code for the 3' part of the gene, i.e., the thrombin-sensitive region, the four domains that are homologous to the epidermal growth factor (EGF) precursor, the COOH-terminal part of protein S that is homologous to a plasma sex hormone binding globulin (SHBG), and, finally, the 3' untranslated region. The thrombin-sensitive region and the EGF-like domains are each coded on a separate exon. The sizes of the exons coding for the COOH-terminal half of protein S and the location of the introns are nearly identical with those in the homologous SHBG gene. Furthermore, the phase class of the splice junctions is the same in these two genes. We have also isolated and mapped genomic clones that cover 25 kilobases of the protein S beta-gene, which was found to contain stop codons and a 2 bp deletion which introduces a frame shift, suggesting that it is a pseudogene. The structure of the two protein S genes and a comparison with the vitamin K dependent clotting factors support a model for their origin by exon shuffling and recruitment of the 3' part of the gene from an ancestor shared with the sex hormone binding globulin.

Criteria for detection of deep venous thrombosis by 99Tcm-plasmin test.

Criteria for the interpretation of 99Tcm-plasmin test results were systematically investigated in 353 patients with suspected deep venous thrombosis (DVT). Scintillation detector measurements were made at 12 points on each leg after intravenous injection of 99Tcm-porcine plasmin. Phlebography was used as a reference method. The criteria were chosen to obtain the highest possible sensitivity (98 to 100%). On this assumption, the specificity reached 66 to 69%, when using the two most efficient criteria. These criteria involved a comparison between count rates at three adjacent points of the leg suspected of having DVT, and count rates at the three corresponding points of the other leg. Comparison between measurements performed at 5 and 30 min after the injection showed a higher sensitivity of the test at the 30 min measurement. The specificity decreased when the results from both 5 and 30 min measurements were combined. Further increase in specificity could only be achieved at the cost of decreased sensitivity. The suggested criteria seem to be the most efficient for this kind of radionuclide test and improved test-efficiency has to await more thrombus-specific radiopharmaceuticals.

Follow-up of circulatory changes secondary to deep venous thrombosis with special regards to radionuclide tests.

The influence of circulatory changes, which are secondary to deep venous thrombosis (DVT) in the leg, on result of radionuclide tests was studied in eight patients. Strain gauge plethysmography, a radionuclide blood-pool test and phlebography were performed both in the acute phase and during recovery up to 6 months after the initial admission. Morphological and functional changes were correlated with results from repeatedly performed 99Tcm-plasmin tests, a test currently used for diagnosis of DVT. In the acute phase, the thrombotic leg showed an increase in pooled blood and, in the case of proximal thrombosis, also impaired venous outflow. During the 6-month follow-up complete recanalization was observed in three patients and partial recanalization in five. The circulatory changes were found to recover progressively and earlier than the morphological changes. The 99Tcm-plasmin test was pathological at admission in all patients. It was normalized in parallel with plethysmography and blood-pool test results, at a time when morphological recovery was still incomplete. These findings confirm that a positive 99Tcm-plasmin test reflects haemodynamic changes which are secondary to the DVT rather than a specific binding of the radiopharmaceutical to the thrombus. The 99Tcm-plasmin test was normalized from 1 to more than 26 weeks after an acute DVT. This finding is of practical importance when using radionuclide tests for evaluation of acute recurrent DVT.

Return to normal of 99mTc-plasmin test after deep venous thrombosis and its relationship to vessel wall fibrinolysis.

Fourteen patients with deep venous thrombosis (DVT) and a positive 99mTc-plasmin test were followed up to determine how soon a negative test was obtained. Localization and extension of the thrombi were determined by phlebography. Plasminogen activator activity in vein walls and local fibrinolytic activity after venous occlusion were measured in order to find out what the prerequisites for impaired thrombolysis are. The time required to obtain a negative 99mTc-plasmin test showed considerable variation, ranging from less than 1 week to more than 6 months. The 99mTc-plasmin test had returned to normal in 64% of the patients after 6 months. No relationship was found between vessel wall fibrinolysis and time to normalization. Instead, we found an association between the time to normalization of the 99mTc-plasmin test and the size of the thrombus, according to phlebography, as well as between the time to normalization of the 99mTc-plasmin test and the extension of leg points with a positive 99mTc-plasmin test at admission. The finding of abnormal 99mTc-plasmin test results more than 6 months after acute DVT is of practical importance and warrants caution when evaluating patients with symptoms and signs suggestive of acute recurrent DVT.

Quantitative in vivo and in vitro comparison between radiolabelled colloids, biomolecules and blood cells in their ability to diagnose deep venous thrombosis.

Three radiopharmaceutical groups (colloids, specific biomolecules and blood cells) and a control group were investigated with regard to their ability to rapidly diagnose deep venous thrombosis in an experimental rabbit model. An artificial thrombus was induced in the jugular vein and the radiopharmaceuticals were injected either homolaterally or contralaterally relative to the thrombus. The accumulation of the radioactivity in the thrombus 30 min after the induction was determined in vivo from scintillation camera images. After dissection of the jugular vein, the radioactivity of the thrombus was measured in vitro. None of the investigated radiopharmaceutical groups showed any marked high thrombus uptake after contralateral injections, not even the groups that consisted of substances known to be actively involved in coagulation and fibrinolysis. The results exclude a high degree of specific interaction between the radiopharmaceuticals and the thrombus in our model. After homolateral injection only colloids and reduced 99Tcm-pertechnetate showed a high thrombus uptake, thus also excluding a specific binding to thrombus. This investigation shows that none of the specific radiopharmaceuticals had a greater ability to accumulate in the thrombus than the colloids, and it is therefore suggested that the clinical usefulness is due to other mechanisms, like circulatory changes secondary to the DVT.

Comparison between 99Tcm-porcine plasmin and 99Tcm-labelled erythrocytes in diagnosis of deep vein thrombosis.

In 20 patients with suspect deep venous thrombosis (DVT), scintillation detector measurements were performed over each leg during the first 60 min after intravenous injection of 99Tcm-porcine plasmin. Thereafter, 99Tcm-labelled autologous erythrocytes were injected i.v. and repeat measurements were performed. Finally, scintillation camera images of both legs were obtained. Phlebography was used as a reference method. A close relationship (P less than 0.01) was found between the scintillation detector measurements, both in patients with DVT (n = 11) and in patients without DVT (n = 9). Thus, 99Tcm-plasmin is not specifically bound to the thrombus. Rather the clinical utility of the test depends mainly on circulatory changes secondary to the thrombus. Scintillation camera images of 99Tcm-erythrocytes in the legs were not useful for diagnosis of DVT in the calves but showed a high specificity for DVT in the popliteal and femoral veins.

Diagnosis of deep venous thrombosis by 99mTc-human serum albumin microcolloid.

99mTc-human serum albumin microcolloid (HSAC) was evaluated for diagnosis of deep venous thrombosis (DVT) in the legs of 38 consecutive patients. Five and thirty minutes after IV injection, the 99mTc-HSAC activity in both legs was measured by external counting using a collimated NaI (T1)-detector. The relative predominance of 99mTc-HSAC activity in the diseased leg was calculated. Phlebography was performed as a control after the 99mTc-HSAC test. In our hands the non-invasive 99mTc-HSAC test appeared to be rapid, easy to perform and convenient to the patient. The test showed a high sensitivity (11/13) and a low specificity (10/20) compared with phlebography. No adverse reactions were found. The results seem promising and further studies are in progress to establish the value of the 99mTc-HSAC test in screening for DVT.

Diagnosis of deep venous thrombosis by phlebography and 99Tcm-plasmin.

One hundred and thirty-four patients admitted to the medical emergency ward due to suspect deep venous thrombosis (DVT) were examined. The uptake of intravenously injected porcine 99Tcm-plasmin was estimated in both legs. Thereafter, phlebography was performed using a high osmolar contrast medium. All phlebographies were evaluated independently. All patients with negative phlebography were examined clinically after 3-5 days. The plasmin test and phlebography were repeated when called for. The sensitivity of the plasmin test was 100% and the specificity 51% when compared to phlebography. The extension of the DVT as demonstrated by the plasmin test was similar to that determined by phlebography. Post-phlebographic thrombosis was very rare. It is concluded that 99Tcm- plasmin test is a rapid method, convenient to the patient and well suitable as a screening test. The results indicate that a negative plasmin test excludes DVT while a positive test necessitates additional examination by phlebography.