Patent Application: US-88344705-A

Abstract:
the present invention relates to a diagnosis maker for a cardiovascular disease comprising platelet - derived microparticles . the present invention also relates to a method for diagnosing a cardiovascular disease in a subject with symptoms of cardiovascular disease or suspected of having cardiovascular disease , wherein said method comprises : obtaining a sample from said subject ; reacting an antibody to platelet - derived microparticles or a fragment thereof with a biological sample ; detecting a presence or an absence of the platelet - derived microparticles or a fragment thereof in said sample ; and diagnosing cardiovascular disease in said subject having said symptoms when said platelet - derived microparticles or a fragment thereof are detected in said sample .

Description:
the present invention relates to a novel diagnosis maker for a cardiovascular disease comprising platelet - derived microparticles . inflammation as well as platelet activation at the site of local vessel - wall injury plays an essential role in the mechanism of restenosis after percutaneous coronary intervention ( pci ). platelet - derived microparticles ( pdmps ) released from activated platelets are thought to play a role in the inflammatory process , interacting with leukocyte integrin mac - 1 . the present inventors serially measured circulating pdmps by elisa in 35 patients undergoing coronary stenting . inventors also investigated serial changes in high - sensitive c - reactive protein ( hs - crp ) and activated mac - 1 on the surface of neutrophils . pdmps , hs - crp and activated mac - 1 increased after coronary stenting in a time - dependent manner with the maximum response at 48 hr in coronary sinus blood ( pdmps : 10 . 2 ± 5 . 7 to 30 . 4 ± 14 . 6 u / ml ; p & lt ; 0 . 001 , hs - crp : 0 . 26 ± 0 . 22 to 1 . 51 ± 0 . 88 mg / dl ; p & lt ; 0 . 001 , activated mac - 1 , 138 ± 17 % relative increase , p & lt ; 0 . 001 ). these changes were less striking in the peripheral blood . the value of pdmps was correlated with hs - crp levels ( r = 0 . 58 , p & lt ; 0 . 001 ) and the relative increase in activated mac - 1 ( r = 0 . 69 , p & lt ; 0 . 001 ) in coronary sinus blood 48 hrs after coronary stenting . the pdmp values , hs - crp levels and relative increase in activated mac - 1 at 48 hr after coronary stenting were all associated with angiographic late lumen loss . these results suggest that coronary stenting enhanced circulating pdmps in association with an inflammatory response in the injured vessel wall . pdmps may be a useful marker for evaluation of stent - induced inflammatory status and a sound surrogate marker for activated mac - 1 . platelet - derived microparticles ( pdmp ( s )) refer to microparticles released from platelets in association with platelet activation , the contents of which include platelet granular proteins such as p - selectin , and various platelet surface membrane glycoproteins such as glycoprotein ( gp ) ib / ix or gpiib / iiia 1 . pdmps are not merely a marker for platelet activation but also have pro - coagulant activity , and thereby , contribute to thrombus formation 2 . in addition , pdmps participate in the inflammatory process as a mediator of platelet - leukocyte , leukocyte - endothelial cell or leukocyte - leukocyte interactions 1 . pdmps stimulate cytokine production and enhance the expression of cell adhesion molecules including leukocyte integrin mac - 1 ( cd11b / cd18 , αmβ2 ) 3 . although pdmps are usually determined by flow cytometry , circulating pdmps are also measured by enzyme - linked immunosorbent assay ( elisa ) using two antibodies against the platelet membrane surface glycoproteins , gpib and gbix 4 . in the present invention , the inventors demonstrated that circulating levels of pdmps , hs - crp levels , and 8b2 binding on the surface of neutrophils increased in a time - dependent manner after coronary stenting with the maximum increase at 48 hr . furthermore , these changes were more striking in the coronary sinus than in the peripheral blood and the pdmp levels at 48 hr were correlated with the hs - crp levels at 48 hr and more closely with increase in 8b2 binding at 48 hr versus baseline in the coronary sinus . in addition , these three parameters were associated with angiographic late lumen loss ( i . e ., neo - intimal thickening after coronary stenting ). these results indicated that the pdmps increased in the coronary circulation , and were associated with mac - 1 activation on the surface of neutrophils in relation to stent - induced inflammatory response , possibly at sites of pci - induced injury . the cardiovascular disease includes , but are not limited to , arterial hypertension , orthostatic hypotension and syncope , arteriosclerosis , coronary artery disease , heart failure , shock , arrhythmias , cardiac and respiratory arrest and cardiopulmonary resuscitation , valvular heart disease , endocarditis , pericardial disease , cardiac tumors , diseases of the aorta and its branches , peripheral vascular disorders , cerebrovascular disease , diabetic vascular disorders , or hyperlipidemia , and the like , and also the high risk group of potentially having these disorders . arteriosclerosis is mostly preferred for the present invention . detection for a diagnosis of cardiovascular disease , or an evaluation of prognosis or progress of cardiovascular disease is performed by immunologic assays using antibodies to pdmps or a fragment thereof and samples from subjects in need of such diagnosis . thus the method of the invention includes methods that detect pdmps in a sample , for example , in a non - specific manner or in a specific manner , and then detecting such non - specific or specific binding . the above examples of methods are not the only methods by which one skilled in the arts of medicine , microbiology , and immunology may use the invention . any method that allows determination of an interaction of the immune system with pdmps in a sample is useful in the method of the invention . according to the method of the invention , a diagnosis of cardiovascular disease in a patient suspected of having such disease , or an evaluation of the severity or progression of cardiovascular disease in a patient already diagnosed as having the same , is made by detecting an immune response against pdmps in a sample from the patient in need of such diagnosis . when it is desired to detect pdmps , generally and preferably , pdmps in the patient &# 39 ; s sample are detected . the detection of pdmps can be performed by directly detecting the binding of such antibodies to pdmps . alternatively , the detection of antibodies that bind to pdmps can be performed by indirectly detecting the binding of such antibodies to pdmps . the term “ antibodies ” in meant to include both the native antibodies , and biologically active derivatives of antibodies , such as , for example , fab ′, f ( ab ′) 2 or fv as well as single - domain and single - chain antibodies . a biologically active derivative of an antibody retains the ability to bind an antigen . the pdmps in the patient &# 39 ; s sample , and especially in blood , serum , cell or tissue sample , can be detected in immunoassays wherein the pdmps can be utilized in liquid phase or bound to a solid phase carrier . the preferred immunoassays for detecting pdmps using the methods of this invention include radioimmuno - assays , enzyme - linked immunosorbent assays ( elisa ), or other assays known in the art , such as immunofluoscent assays , chemiluminescent assays , or bioluminescent assays . the preferred immunoassay for detecting the pdmps is elisa assay . pdmps have been widely measured by flow cytometry and investigated extensively . although the significance of elevated pdmp levels remains controversial , many clinical disorders are associated with elevated pdmp levels 1 , 2 , 21 - 24 . elevated pdmps are observed in patients with acute coronary syndrome ( acs ) in relation to platelet activation 23 . gawaz et al . 24 examined various aspects of platelet activation in patients with acute myocardial infarction undergoing direct pci and demonstrated that pdmps were significantly enhanced after pci . however , pdmps measure by elisa , as in the present invention , may have different clinical features from those detected by flow cytometry . nomura et al 26 observed serial changes in pdmps measured by elisa after pci in acs patients and demonstrated that pdmp levels decreased significantly 4 days after pci . their results are contrary to ours that showed increased pdmps with the maximum increase 48 hr after pci . this discrepancy may be attributed to differences in the present invention populations . the study by nomura et al . selected only acs patients whose platelets had already been activated before pci and the activation status was stabilized 4 days after pci , while the present invention included patients with stable angina alone without platelet activation at baseline before pci . the time course of pdmp changes that inventors observed indicated that stable platelets were activated by pci with the maximum activation at 48 hr , which is consistent with the time course of p - selectin changes post - pci that has been previously reported by us as well as others . the activation of leukocytes , neutrophils and monocytes is known to play an important causative role in the post - pci inflammatory process that leads to the development of restenosis 27 - 30 . activated leukocytes transmigrate and infiltrate into the pci - injured vessel wall and produce various cytokines , growth factors , free radicals , and proteolytic enzymes , leading to neointimal thickening and restenosis . at the pci - injured vessel wall , which is denuded of vascular endothelial cells by balloon inflation or stenting , platelets first adhere to the vessel surface and the platelet layer is formed . leukocytes adhere to the platelet layer and then migrate into the vessel wall — namely , transplatelet leukocyte migration . 9 , 31 , 32 in the process of transplatelet leukocyte migration , platelet surface p - selectin binds to p - selectin glycoprotein ligand ( psgl )- 1 on the surface of leukocytes and mediates the rolling attachment of leukocytes with the platelet layer . 33 , 34 in addition , subsequent firm adhesion of leukocytes is mediated by mac - 1 , which is expressed on activated leukocytes and binds to ligands such as fibrinogen 9 , 33 , gpibα 10 , intercellular adhesion molecules ( icam )- 2 9 , or junctional adhesion molecules ( jam )- 3 35 . among these platelet ligands for mac - 1 binding , simon et al . especially focused on gpibα as the most important ligand in the mechanism of transplatelet leukocyte migration in the vessel wall injured by pci . evangelista et al . 33 , 34 demonstrated in - vitro that the binding of p - selectin to psgl - 1 triggers tyrosine kinase - dependent signaling that leads to functional up - regulation or activation of mac - 1 . in this way , an adhesion cascade appears to occur with considerable crosstalk between p - selectin and mac - 1 in the process of platelet - leukocyte interaction . 9 , 33 moreover , forlow et al . 36 reported that p - selectin - expressing pdmps bind to leukocytes that express psgl - 1 , suggesting that pdmps can mediate leukocyte - leukocyte interaction leading to leukocyte aggregation and accumulation at the injured surface of the vessel surface , especially when the number of pdmps increased . thus , the measurement of pdmps may be useful for investigating this pathophysiological process . in addition , in the pdmp elisa assay system , the inventors used an antibody against gpibα for detecting pdmps 4 . considering that gpibα is the key ligand for mac - 1 in transplatelet leukocyte migration , pdmps detected by elisa may be a surrogate for leukocyte mac - 1 activity . our clinical finding of a close correlation between pdmps at 48 hr and the increase over baseline in activated mac - 1 on the surface of neutrophils at 48 hr supports this hypothesis . the present invention has several potential limitations . although pdmps are not the only products of platelet activation but also their own function such as procoagulant activity and participants in the inflammatory process as a mediator of platelet - leukocyte interaction , circulating pdmps detected by elisa assay are likely to be a residue of activated pdmps that adhere to leukocytes . 4 therefore , assigning a pathophysiological role to pdmps in the vascular injury , inflammation and repair response remains speculative , but highly likely in light of our observations showing the relationship between pdmps , crp , and mac - 1 activity after pci or the relationship between those and angiographic late lumen loss . furthermore , pdmps , easily measured by elisa , may serve as a useful surrogate of activated mac - 1 that can only be measured with a complex technique such as flow cytometry . restenosis , the most significant problem with pci , has been markedly reduced since the introduction of coronary stents . recent advances in drug - eluting stents have further reduced the restenosis rate to less than 10 %. however , even drug - eluting stents are not perfect and have several serious problems such as long - term prognosis or late incomplete apposition . thus , the issue of restenosis has not been completely resolved and the inventors should continue to develop approaches to further reduce restenosis . recent chemical , biological or pharmacological approaches to prevent restenosis include two strategies , an ‘ anti - proliferative ’ strategy and an ‘ anti - inflammatory ’ strategy . most novel approaches for reducing restenosis , including newly developed drug - eluting stents , have employed an ‘ anti - proliferative ’ strategy . for further restenosis reduction , the inventors have proposed an ‘ anti - inflammatory strategy ’, which appears to be a rational therapeutic strategy for preventing restenosis . in addition , the inventors can envision a significant clinical advantage of mac - 1 - guided therapy for reducing restenosis , in which pdmps measured by elisa would serve as a sound surrogate marker for activated mac - 1 . the subjects included 35 patients with atherosclerotic coronary artery disease who underwent single elective coronary stent implantation for a proximal left anterior descending artery ( lad ) lesion . the patients &# 39 ; characteristics are shown in table 1 . to reduce the heterogeneity of the population , the inventors excluded patients with poorly - controlled diabetes mellitus , hypertension or hyperlipidemia , or with a systemic inflammatory reaction as shown by a baseline crp & gt ; 1 . 5 mg / dl . all of the patients received standard daily oral medications for angina , including 81 mg of aspirin , and none of these medications were discontinued or exchanged during pci or the post - pci follow - up period . the patients received 200 mg of daily oral ticlopidine 2 days before pci as a specific post - stent anti - platelet regimen and this therapy was continued for one month after pci . coronary stent implantation was performed using the standard judkins technique from a femoral approach . intravenous heparin was administered to maintain an adequate activated clotting time ( act ) during the procedure and for 48 hrs after pci . follow - up angiography was recommended for all patients at 6 months after angioplasty , and was performed earlier if clinically indicated . coronary lesions were assessed by quantitative coronary angiographic ( qca ) measurements and late lumen loss ( minimal lumen diameter after pci minus minimal lumen diameter at follow - up angiography ) was calculated as an index of neointimal thickening . prior to pci , a coronary sinus catheter was positioned in the coronary sinus and left for 48 hr after the procedure for coronary sinus blood sampling . coronary sinus blood as well as peripheral blood was collected before pci and 15 min after , 24 hr after , and 48 hr after coronary stenting . whole blood was immediately collected into tubes containing acid citrate dextrose ( acd ), ethylene diaminetetraacetate ( edta ), or both ( acd / edta ). the study protocol was approved by the local institutional review board , and written informed consent was obtained from each patient . the acd / edta blood was centrifuged at 5000 × g for 20 min , plasma was withdrawn and stored at − 80 ° c . until assay . the assay for circulating pdmps was performed using enzyme - linked immunosorbent assay ( elisa ), as previously reported . 4 briefly , 50 μl of samples or standard were added to each well of 96 - well microtiter plates coated with antibody against platelets and gpxi ( mkp - 9 ) and incubated for 18 hrs at 25 ° c . on a plate shaker ( 200 rpm ). plates were washed 3 times with 350 μl / well of buffer ( 0 . 05 % tween 20 in pbs ). fifty μl of biotinylated antibody against platelet gpibα ( nnky5 - 5 ) ( 0 . 2 μl / ml in 1 % nonfat milk / pbs ) was added to each well and incubated for 2 hrs at 25 ° c . on a plate shaker . after each well was washed 3 times with 350 μl of buffer , 50 μl of peroxidase - conjugated avidin ( diluted 1 : 20000 in 1 % nonfat dry milk , pbs ; vector laboratories , burlingame , calif .) was added to each well and incubated for 2 hrs at 25 ° c . on a plate shaker . each well was subsequently washed 3 times with 350 μl of buffer and then incubated with 100 μl peroxidase substitute solution ( scytek , logan , utah ) for 20 min at room temperature . after this incubation , stop solution ( skytec ) was added to each well , and the absorbance was measured with an elisa reader at 450 nm . the edta blood was centrifuged at 1 , 500 × g for 15 min at room temperature for the measurement of high - sensitive ( hs )- crp . the plasma was frozen and stored at − 80 ° c . until analysis . the hs - crp level was measured by particle - enhanced technology on the behring bn ii nephelometer ( dade behring inc ., newark , del .) 19 . this assay used monoclonal anti - crp antibodies and a calibrator that was also traceable to who reference material . the acd whole blood was used for flow cytometric analysis for expression of activation - dependent neoepitope of mac - 1 on the surface of neutrophils . the inventors used a purified monoclonal antibody , 8b2 ( provided by dr . thomas edgington , department of immunology , the scripps research institute , la jolla , calif . ), with a high sensitivity and specificity for the recognition of the activation - dependent neo - epitope of mac - 1 18 , 20 . purified mouse immunoglobulin ( ig ) g1 was also used as an isotype - negative control . the fluorescein - conjugated second - step reagents for indirect immunofluorescence were fluorescein isothiocyanate ( fitc )- conjugated f ( ab ′) 2 fragment of anti - mouse igg goat immunoglobulins ( dako , glostrup , denmark ). indirect immunofluorescence labeling was performed on whole blood incubated with 8b2 ( 100 μg / ml ). then , the flow cytometric analysis for 8b2 binding ( activated mac - 1 ) was performed using an epics xl flow cytometer ( coultronics , sunnyvale , calif .). mean channel fluorescence intensity ( mfi ) was calculated as an index of activated mac - 1 on the surface of neutrophils . values were expressed as the mean ± sd . in both studies , inter - group comparisons were performed using student &# 39 ; s unpaired t test for continuous variables . serial changes in the variables were evaluated by repeated measures analysis of variance ( anova ), for intra - and inter - group comparisons . correlations between two parameters were assessed using simple linear regression . p & lt ; 0 . 05 was considered to be significant . baseline circulating pdmps , hs - crp and mfi for 8b2 binding before pci in the peripheral blood was independent of age , gender , the presence of multivessel disease , coronary risk factors , or medications possibly affecting inflammation such as statins or angiotensin receptor blockers ( table 2 ). serial change in circulating pdmps , hs - crp levels , and activated mac - 1 on the surface of neutrophils after coronary stenting serial changes in circulating levels of pdmps were observed . although no change was evident 15 min after stenting , an increase was noted after 24 hr in both coronary sinus and peripheral blood . the maximal increase was seen at 48 hr ( coronary sinus : 10 . 2 ± 5 . 7 to 30 . 4 ± 14 . 6 u / ml , p & lt ; 0 . 001 ; peripheral blood : 8 . 8 ± 6 . 2 to 22 . 6 ± 8 . 4 u / ml , p & lt ; 0 . 001 ). the changes were more striking in coronary sinus blood than in peripheral blood ( p & lt ; 0 . 05 ) ( fig1 , left ). the plasma hs - crp levels also increased after coronary stenting from baseline values in the same manner as the pdmps , reaching a maximum at 48 hrs in both coronary sinus blood ( 0 . 26 ± 0 . 22 to 1 . 51 ± 0 . 88 mg / dl , p & lt ; 0 . 001 ) and peripheral blood ( 0 . 22 ± 0 . 21 to 1 . 22 ± 0 . 49 mg / dl , p & lt ; 0 . 001 ). the changes in hs - crp levels were also more striking in coronary sinus blood than in peripheral blood ( p & lt ; 0 . 05 ) ( fig1 , mid ). the mfi for 8b2 binding ( i . e ., expression of mac - 1 activation neoepitope on the surface of neutrophils ) began to increase 15 min after coronary stenting and reached a maximum at 48 hr in both coronary sinus and peripheral blood . the relative increases compared to baseline at 15 min , 24 hr and 48 hr in coronary sinus blood were 108 ± 12 % ( p & lt ; 0 . 05 ), 119 ± 11 % ( p & lt ; 0 . 01 ) and 138 ± 17 % ( p & lt ; 0 . 001 ), respectively . the increases in peripheral blood were 106 ± 8 % ( ns ), 114 ± 8 % ( p & lt ; 0 . 05 ) and 127 ± 12 % ( p & lt ; 0 . 01 ), respectively . the change in 8b2 binding capacity was also more striking in coronary sinus blood than in peripheral blood ( p & lt ; 0 . 05 ) ( fig1 , right ). the relation between pdmps , hs - crp and activated mac - 1 at 48 hr the circulating pdmps at 48 hr were positively correlated with hs - crp levels at 48 hr ( r = 0 . 58 , p & lt ; 0 . 001 ) and more closely correlated with the relative increase in 8b2 binding on the surface of neutrophils at 48 hr compared to baseline ( r = 0 . 69 , p & lt ; 0 . 00 ) in coronary sinus blood ( fig2 ). the relation between late lumen loss and pdmps , bs - crp and activated mac - 1 at 48 hr using the median values of pdmps ( 26 u / ml ), hs - crp ( 1 . 3 mg / dl ) and the relative increase in 8b2 binding ( 130 %) at 48 hr in all of the patients , the patients were divided into two groups for each parameter . the high - value group included values 2 than the median while the low - value group included values & lt ; than the median . angiographic late lumen loss was greater in the high - value group than in the low - value group for pdmps ( 1 . 22 ± 0 . 67 vs 0 . 62 ± 0 . 64 mm , p & lt ; 0 . 05 ), hs - crp ( 1 . 26 ± 0 . 71 vs 0 . 49 ± 0 . 95 mm , p & lt ; 0 . 05 ) and the increase in 8b2 binding ( 1 . 42 ± 0 . 39 vs 0 . 44 ± 0 . 76 mm , p & lt ; 0 . 05 ) ( fig3 ). 2 sims p j , faioni e m , wiedmer t , et al . complement proteins c5b - 9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor va and express prothrombinase activity . j biol chem 1988 ; 263 : 18205 - 18212 . 3 barry o p , pratico d , savani r c , et al . modulation of monocyte - 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